Stable and soluble antibodies inhibiting vegf

ABSTRACT

The present invention relates to soluble and stable anti-VEGF immunobinders comprising CDRs from rabbit monoclonal antibodies. Said antibodies are designed for the diagnosis and/or treatment of VEGF-mediated disorders. The hybridomas, nucleic acids, vectors and host cells for expression of the recombinant antibodies of the invention, methods for isolating them and the use of said antibodies in medicine are also disclosed.

RELATED INFORMATION

The present application is a 371 application, which claims priority fromPCT/CH2009/000220, of 25 Jun. 2009, which claims priority to U.S.61/133,212 filed on Jun. 25, 2008, to U.S. 61/075,697 of 25 Jun. 2008,to U.S. 61/155,041 of 24 Feb. 2009, and to U.S. 61/075,692 of 25 Jun.2008.

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

BACKGROUND OF THE INVENTION

Angiogenesis is implicated in the pathogenesis of a variety of disordersincluding solid tumors, intraocular neovascular syndromes such asproliferative retinopathies or age-related macular degeneration (AMD),rheumatoid arthritis, and psoriasis (Folkman et al. J. Biol. Chem.267:10931-10934 (1992); Klagsbrun et al. Annu Rev. Physiol. 53:217-239(1991); and Garner A, Vascular diseases. In: Pathobiology of oculardisease. A dynamic approach. Garner A, Klintworth G K, Eds. 2nd EditionMarcel Dekker, NY, pp 1625-1710 (1994)). In solid tumors, angiogenesisand growth of new vasculture permits survival of the tumor, and acorrelation has been demonstrated between the density of microvessels intumor sections and patient survival in breast and other cancers (Weidneret al. N Engl J Med 324:1-6 (1991); Horak et al. Lancet 340:1120-1124(1992); and Macchiarini et al. Lancet 340:145-146 (1992)).

Vascular endothelial growth factor (VEGF) is a known regulator ofangiogenesis and neovascularization, and has been shown to be a keymediator of neovascularization associated with tumors and intraoculardisorders (Ferrara et al. Endocr. Rev. 18:4-25 (1997)). The VEGF mRNA isoverexpressed in many human tumors, and the concentration of VEGF in eyefluids are highly correlated to the presence of active proliferation ofblood vessels in patients with diabetic and other ischemia-relatedretinopathies (Berkman et al., J Clin Invest 91:153-159 (1993); Brown etal. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res. 53:4727-4735(1993); Mattern et al. Brit. J. Cancer. 73:931-934 (1996); and Dvorak etal. Am J. Pathol. 146:1029-1039 (1995); Aiello et al. N. Engl. J. Med.331:1480-1487 (1994)). In addition, recent studies have shown thepresence of localized VEGF in choroidal neovascular membranes inpatients affected by AMD (Lopez et al. Invest. Ophtalmo. Vis. Sci.37:855-868 (1996)). Anti-VEGF neutralizing antibodies can be used tosuppress the growth of a variety of human tumor cell lines in nude miceand also inhibit intraocular angiogenesis in models of ischemic retinaldisorders (Kim et al. Nature 362:841-844 (1993); Warren et al. J. Clin.Invest 95:1789-1797 (1995); Borgstrom et al. Cancer Res. 56:4032-4039(1996); and Melnyk et al. Cancer Res. 56:921-924 (1996)) (Adamis et al.Arch. Opthalmol. 114:66-71 (1996)).

Thus, there is a need for anti-VEGF monoclonal antibodies capable ofbeing used for the treatment of solid tumors and various neovascularintraocular diseases.

SUMMARY OF THE INVENTION

The invention provides soluble and stable anti-VEGF immunobinderscomprising CDRs from rabbit monoclonal antibodies. Said antibodies aredesigned for the diagnosis and/or treatment of VEGF-mediated disorders.The hybridomas, nucleic acids, vectors and host cells for expression ofthe recombinant antibodies of the invention, methods for isolating themand the use of said antibodies in medicine are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the binding kinetics of selected scFvs to hVEGF₁₆₅using Biacore (hVEGF165). FIG. 1 a shows the data obtained for 511max:Ka (1/Ms): 6.59E+05; SE (ka): 1.10E+03; kd(l/s):4.40E-05;SE(kd):6.30E-07; KD(M): 6.67E-11. FIG. 1 b shows the data obtained for578max: Ka (1/Ms): 7.00E+05; SE (ka): 1.40E+03; kd(l/s): 3.07E-04;SE(kd): 8.50E-07; KD(M): 4.39E-10.

FIG. 2 illustrates the species specificity by showing binding kineticsof 578max to human, mouse and rat VEGF. FIG. 2 a shows the data obtainedfor human VEGF165: Ka (1/Ms): 7.00E+05; SE (ka): 1.40E+03; kd(l/s):3.07E-04; SE(kd): 8.50E-07; KD(M): 4.39E-10. FIG. 2 b shows the dataobtained for mouse VEGF164: Ka (1/Ms): 1.03E+06; SE (ka): 2.30E+03;kd(l/s): 4.40E-04; SE(kd): 9.40E-07; KD(M): 4.29E-10. FIG. 2 c shows thedata obtained for rat VEGF164: Ka (1/Ms): 8.83E+05; SE (ka): 2.50E+03;kd(l/s): 5.28E-04; SE(kd): 1.20E-06; KD(M): 5.98E-10.

FIG. 3 illustrates the binding kinetics of 578max to VEGF isoforms(hVEGF121 and hVEGF110). FIG. 3 a shows the data obtained for humanVEGF165: Ka (1/Ms): 7.00E+05; SE (ka): 1.4E+03; kd(l/s): 3.07E-04;SE(kd): 8.50E-07; KD(M): 4.39E-10. FIG. 3 b shows the data obtained forhuman VEGF121: Ka (1/Ms): 5.87E+05; SE (ka): 1.20E+03; kd(l/s):5.58E-04; SE(kd): 9.60E-07; KD(M): 9.50E-11. FIG. 3 c shows the dataobtained for human VEGF110: Ka (1/Ms): 5.23E+05; SE (ka): 1.30E+03;kd(l/s): 7.22E-04; SE(kd): 8.10E-07; KD(M): 1.38E-09.

FIG. 4 depicts the binding kinetics of 578max, 578minmax and 578 wt tohVEGF165. FIG. 4 a shows the data obtained for 578max: Ka (1/Ms):7.00E+05; SE (ka): 1.40E+03; kd(l/s): 3.07E-04; SE(kd): 8.50E-07; KD(M):4.39E-10. FIG. 4 b shows the data obtained for 578minmax: Ka (1/Ms):8.06E+05; SE (ka): 2.10E+03; kd(l/s): 5.04E-04; SE(kd): 1.10E-06; KD(M):6.25E-10. FIG. 4 c shows the data obtained for 578 wt-His: Ka (1/Ms):8.45E+05; SE (ka): 1.60E+03; kd(l/s): 1.69E-04; SE(kd): 7.60E-07; KD(M):2.00E-10.

FIG. 5 illustrates thermal stability of 578max, 578minmax and578minmax_DHP (unfolding measured by FT-IR). FIG. 5 a: 578minmax(ESBA903): Tm=71.1° C.; FIG. 5 b: 578minmax_DHP (#961): Tm=70.2° C.;FIG. 5 c: 578max (#821): Tm=70.4° C.

FIG. 6 illustrates denaturation and precipitation of 578 derivativesafter thermal stress (FIG. 6 a: 50° C., FIG. 6 b: 60° C., FIG. 6 c: 70°C.) for 30 min.

FIG. 7 illustrates solubility of 578max, 578minmax and 578minmax_DHP(determined by ammonium sulfate precipitation). FIG. 7 a: 578max (#821).The V50 was 27.24% FIG. 7 b: 578minmax (ESBA903). The V50 was 28.13.FIG. 7 c: 578minmax_DHP (#961). The V50 was 32.36%.

FIG. 8 illustrates VEGFR2 competition ELISA versus HUVEC assay asmethods to measure potency. FIG. 8 a: Comparison of Lucentis and 511max(#802) in VEGFR2 competition ELISA. R² of Lucentis: 0.9417; R² ofESBA802: 0.9700. EC50 of Lucentis: 7.137 nM; EC50 of #802: 0.8221 nM.FIG. 8 b: Comparison of Lucentis and 578max (#821). in VEGFR2competition ELISA. FIG. 8 c: Comparison of Lucentis, 511maxC-his and534max in HUVEC assay. R² of Lucentis 0.9399; R² of EP511maxC-his:0.9313, R² of EP534max: 0.7391. EC50 of Lucentis: 0.08825 nM, EC50 of511maxC-his: 0.7646 nM, EC50 of 534max: 63.49 nM. FIG. 8 d: Comparisonof Lucentis, 578 min and 578max in HUVEC assay. R² of Lucentis: 0.9419,R² of EP578 min: 0.8886, R² of EP578max: 0.9274. EC50 of Lucentis:0.1529 nM, EC50 of 578 min: 1.528 nM, EC50 of 578max: 0.1031 nM.

FIG. 9 illustrates the effects of 578minmax on HUVEC proliferationinduced by hVEGF165. The parameters of the assay were the following:hVEGF165 concentration: 0.08 nM (3 ng/ml); incubation with VEGF and testitem: 96 h. The EC50 was 0.08959 nM for Lucentis and 0.05516 nM for578minmax, whereas the R² was 0.9066 for Lucentis and 0.9622 for578minmax.

FIG. 10 illustrates the effects of 578minmax on HUVEC Proliferationinduced by mouse VEGF164 and rat VEGF164. The parameters of the assaywere the following: mouse VEGF164 concentration: 0.08 nM (3 ng/ml); ratVEGF164 concentration: 0.3 nM (11.3 ng/ml). Both concentrations wereselected at EC90 for VEGF induced HUVEC proliferation; incubation withVEGF and test item: 96 h. FIG. 10 a illustrates the data obtained formouse VEGF. The EC50 was 0.1196 nM for V1253 and 0.06309 nM for578minmax, whereas the R² was 0.02744 for Lucentis, 0.9348 for V1253 and0.9767 for EP578minmax. Lucentis did not inhibit HUVEC proliferationinduced by mouse VEGF. FIG. 10 b illustrates the data obtained for ratVEGF. The EC50 was 1.597 nM for V1253 and 0.06974 nM for 578minmax,whereas the R² was 00.7664 for V1253 and 0.6635 for 578minmax.

FIG. 11 illustrates efficacy studies using Miles assay in nude guineapigs (part I). The dye almar blue 1 was administered intravenously tonude guinea pigs. One hour after dye injection, a premixture 2 of hVEGF(2.61 nM) and Lucentis, ESBA903 or #802, respectively, was injected intothe skin of the animal 3. One hour after injection of the solutions, theanimals 3 were euthanized and the pelts were collected, cleaned andphotographed digitally using incident and transmitted light. The area ofEvans Blue dye that extravasated into the injection sites was evaluatedusing Image J and the dose-area retention was plotted.

FIG. 12 illustrates efficacy studies using Miles assay in nude guineapigs (part II). FIG. 12 a shows the results obtained for #803 (511max).The EC50 was 5.990 nM and had a statistical spread between 2.060 and17.41 nM whereas the R² was 0.5800. FIG. 12 b shows the results obtainedfor ESBA903 (578minmax). The EC50 was 3.989 and had a statistical spreadbetween 1.456 and 10.93 nM whereas the R² was 0.3920. FIG. 12 c showsthe area of dye leakage for Lucentis. The EC50 could not be calculatedfor Lucentis due to the poor fit of the curve.

FIG. 13 illustrates efficacy studies using modified miles assay in rats(premixed hVEGF165 and 578minmax (ESBA903)). FIG. 13 a illustrates theanti-permeability efficacy of Avastin upon VEGF induced retinal vascularleakage in rats—dose response. Avastin inhibits hVEGF-induced retinalvascular permeability. Premixed before injection. Approximatelyequimolar, 3fold, or 10 fold excess. *p<0.05 (VEGF s. BSA), ** p<0.05(Avastin treated vs. VEGF). FIG. 13 b shows the anti-permeabilityefficacy of ESBA903 upon VEGF induced retinal vascular leakage in rats.Dose response (pre-mixed, ivt). Complete inhibition of hVEGF-inducedretinal vascular permeability by ESBA903. Premixed before injection.Approximately equimolar, 3fold, or 10 fold excess. *p<0.05 (VEGF s.BSA), ** p<0.05 (ESBA903 treated vs. VEGF).

FIG. 14 illustrates efficacy studies using modified miles assay in rats(topical administration of 578minmax (ESBA903)). The anti-permeabilityefficacy of AL-51287 (ESBA903) upon VEGF induced retinal vascularleakage in rats was tested upon topical administration. Five dayspretreatment, 4 drops/day with a 10 ng/ml ESBA903 formulation. *p<0.05(VEGF s. BSA), ** p<0.05 (VEGF vs. AL-51287), ***p=0.060 (AL-51287 vs.AL-52667), ****(VEGF vs. AL-39324); p<0.05 (AL-39324 vs. vehicle refctrl). AL-51287: ESBA903; AL-52657: topical vehicle reference control;AL-39324: small molecule RTK inhibitor.

FIG. 15 illustrates the definition of CDR1 of VH as used herein.

DETAILED DESCRIPTION

The invention provides soluble and stable anti-VEGF immunobinderscomprising CDRs from rabbit monoclonal antibodies. Said immunobindersare designed for the diagnosis and/or treatment of VEGF-mediateddisorders. The hybridomas, nucleic acids, vectors and host cells forexpression of the recombinant antibodies of the invention, methods forisolating them and the use of said antibodies in medicine are alsodisclosed.

DEFINITIONS

In order that the present invention may be more readily understood,certain terms will be defined as follows. Additional definitions are setforth throughout the detailed description.

The term “VEGF” refers to the 165-amino acid vascular endothelial cellgrowth factor, and related 121-, 189-, and 206-amino acid vascularendothelial cell growth factors, as described by Leung et al., Science246:1306 (1989), and Houck et al., Mol. Endocrin. 5:1806 (1991) togetherwith the naturally occurring allelic and processed forms of those growthfactors.

The term “VEGF receptor” or “VEGFr” refers to a cellular receptor forVEGF, ordinarily a cell-surface receptor found on vascular endothelialcells, as well as variants thereof which retain the ability to bindhVEGF. One example of a VEGF receptor is the fms-like tyrosine kinase(fit), a transmembrane receptor in the tyrosine kinase family. DeVrieset al., Science 255:989 (1992); Shibuya et al., Oncogene 5:519 (1990).The flt receptor comprises an extracellular domain, a transmembranedomain, and an intracellular domain with tyrosine kinase activity. Theextracellular domain is involved in the binding of VEGF, whereas theintracellular domain is involved in signal transduction. Another exampleof a VEGF receptor is the flk-1 receptor (also referred to as KDR).Matthews et al., Proc. Nat. Acad. Sci. 88:9026 (1991); Terman et al.,Oncogene 6:1677 (1991); Terman et al., Biochem. Biophys. Res. Commun.187:1579 (1992). Binding of VEGF to the flt receptor results in theformation of at least two high molecular weight complexes, having anapparent molecular weight of 205,000 and 300,000 Daltons. The 300,000Dalton complex is believed to be a dimer comprising two receptormolecules bound to a single molecule of VEGF.

The term “rabbit” as used herein refers to an animal belonging to thefamily of the leporidae.

The term “antibody” as used herein is a synonym for “immunoglobulin.”Antibodies according to the present invention may be wholeimmunoglobulins or fragments thereof, comprising at least one variabledomain of an immunoglobulin, such as single variable domains, Fv (SkerraA. and Pluckthun, A. (1988) Science 240:1038-41), scFv (Bird, R. E. etal. (1988) Science 242:423-26; Huston, J. S. et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-83), Fab, (Fab′)2 or other fragments well knownto a person skilled in the art.

The term “CDR” refers to one of the six hypervariable regions within thevariable domains of an antibody that mainly contribute to antigenbinding. One of the most commonly used definitions for the six CDRs wasprovided by Kabat E. A. et al., (1991) Sequences of proteins ofimmunological interest. NIH Publication 91-3242). As used herein,Kabat's definition of CDRs only apply for CDR1, CDR2 and CDR3 of thelight chain variable domain (CDR L1, CDR L2, CDR L3, or L1, L2, L3), aswell as for CDR2 and CDR3 of the heavy chain variable domain (CDR H2,CDR H3, or H2, H3). CDR1 of the heavy chain variable domain (CDR H1 orH1), however, as used herein is defined by the following residues (Kabatnumbering): It starts with position 26 and ends prior to position 36.This is basically a fusion of CDR H1 as differently defined by Kabat andChotia (see also FIG. 15 for illustration).

The term “antibody framework”, or sometimes only “framework”, as usedherein refers to the part of the variable domain, either VL or VH, whichserves as a scaffold for the antigen binding loops (CDRs) of thisvariable domain. In essence it is the variable domain without the CDRs.

The term “single chain antibody”, “single chain Fv” or “scFv” isintended to refer to a molecule comprising an antibody heavy chainvariable domain (or region; V_(H)) and an antibody light chain variabledomain (or region; V_(L)) connected by a linker. Such scFv molecules canhave the general structures: NH₂-V_(L)-linker-V_(H)-COOH orNH₂-V_(H)-linker-V_(L)-COOH.

As used herein, “identity” refers to the sequence matching between twopolypeptides, molecules or between two nucleic acids. When a position inboth of the two compared sequences is occupied by the same base or aminoacid monomer subunit (for instance, if a position in each of the two DNAmolecules is occupied by adenine, or a position in each of twopolypeptides is occupied by a lysine), then the respective molecules areidentical at that position. The “percentage identity” between twosequences is a function of the number of matching positions shared bythe two sequences divided by the number of positions compared ×100. Forinstance, if 6 of 10 of the positions in two sequences are matched, thenthe two sequences have 60% identity. By way of example, the DNAsequences CTGACT and CAGGTT share 50% identity (3 of the 6 totalpositions are matched). Generally, a comparison is made when twosequences are aligned to give maximum identity. Such alignment can beprovided using, for instance, the method of Needleman et al. (1970) J.Mol. Biol. 48: 443-453, implemented conveniently by computer programssuch as the Align program (DNAstar, Inc.). The percent identity betweentwo amino acid sequences can also be determined using the algorithm ofE. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) whichhas been incorporated into the ALIGN program (version 2.0), using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. In addition, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch (J. Mol.Biol. 48:444-453 (1970)) algorithm which has been incorporated into theGAP program in the GCG software package (available at www.gcg.com),using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

“Similar” sequences are those which, when aligned, share identical andsimilar amino acid residues, where similar residues are conservativesubstitutions for corresponding amino acid residues in an alignedreference sequence. In this regard, a “conservative substitution” of aresidue in a reference sequence is a substitution by a residue that isphysically or functionally similar to the corresponding referenceresidue, e.g., that has a similar size, shape, electric charge, chemicalproperties, including the ability to form covalent or hydrogen bonds, orthe like. Thus, a “conservative substitution modified” sequence is onethat differs from a reference sequence or a wild-type sequence in thatone or more conservative substitutions are present. The “percentagesimilarity” between two sequences is a function of the number ofpositions that contain matching residues or conservative substitutionsshared by the two sequences divided by the number of positions compared×100. For instance, if 6 of 10 of the positions in two sequences arematched and 2 of 10 positions contain conservative substitutions, thenthe two sequences have 80% positive similarity.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not negativelyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative sequence modificationsinclude nucleotide and amino acid substitutions, additions anddeletions. For example, modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions includeones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue in a human anti-VEGF antibody is preferably replaced withanother amino acid residue from the same side chain family. Methods ofidentifying nucleotide and amino acid conservative substitutions whichdo not eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997))

“Amino acid consensus sequence” as used herein refers to an amino acidsequence that can be generated using a matrix of at least two, andpreferably more, aligned amino acid sequences, and allowing for gaps inthe alignment, such that it is possible to determine the most frequentamino acid residue at each position. The consensus sequence is thatsequence which comprises the amino acids which are most frequentlyrepresented at each position. In the event that two or more amino acidsare equally represented at a single position, the consensus sequenceincludes both or all of those amino acids.

The amino acid sequence of a protein can be analyzed at various levels.For example, conservation or variability can be exhibited at the singleresidue level, multiple residue level, multiple residue with gaps etc.Residues can exhibit conservation of the identical residue or can beconserved at the class level. Examples of amino acid classes includepolar but uncharged R groups (Serine, Threonine, Asparagine andGlutamine); positively charged R groups (Lysine, Arginine, andHistidine); negatively charged R groups (Glutamic acid and Asparticacid); hydrophobic R groups (Alanine, Isoleucine, Leucine, Methionine,Phenylalanine, Tryptophan, Valine and Tyrosine); and special amino acids(Cysteine, Glycine and Proline). Other classes are known to one of skillin the art and may be defined using structural determinations or otherdata to assess substitutability. In that sense, a substitutable aminoacid can refer to any amino acid which can be substituted and maintainfunctional conservation at that position.

It will be recognized, however, that amino acids of the same class mayvary in degree by their biophysical properties. For example, it will berecognized that certain hydrophobic R groups (e.g., Alanine, Serine, orThreonine) are more hydrophilic (i.e., of higher hydrophilicity or lowerhydrophobicity) than other hydrophobic R groups (e.g., Valine orLeucine). Relative hydrophilicity or hydrophobicity can be determinedusing art-recognized methods (see, e.g., Rose et al., Science, 229:834-838 (1985) and Corvette et al., J. Mol. Biol., 195: 659-685 (1987)).

As used herein, when one amino acid sequence (e.g., a first V_(H) orV_(L) sequence) is aligned with one or more additional amino acidsequences (e.g., one or more VH or VL sequences in a database), an aminoacid position in one sequence (e.g., the first V_(H) or V_(L) sequence)can be compared to a “corresponding position” in the one or moreadditional amino acid sequences. As used herein, the “correspondingposition” represents the equivalent position in the sequence(s) beingcompared when the sequences are optimally aligned, i.e., when thesequences are aligned to achieve the highest percent identity or percentsimilarity.

As used herein, the term “antibody database” refers to a collection oftwo or more antibody amino acid sequences (a “multiplicity” ofsequences), and typically refers to a collection of tens, hundreds oreven thousands of antibody amino acid sequences. An antibody databasecan store amino acid sequences of, for example, a collection of antibodyV_(H) regions, antibody V_(L) regions or both, or can store a collectionof scFv sequences comprised of V_(H) and V_(L) regions. Preferably, thedatabase is stored in a searchable, fixed medium, such as on a computerwithin a searchable computer program. In one embodiment, the antibodydatabase is a database comprising or consisting of germline antibodysequences. In another embodiment, the antibody database is a databasecomprising or consisting of mature (i.e., expressed) antibody sequences(e.g., a Kabat database of mature antibody sequences, e.g., a KBDdatabase). In yet another embodiment, the antibody database comprises orconsists of functionally selected sequences (e.g., sequences selectedfrom a QC assay).

The term “immunobinder” refers to a molecule that contains all or a partof the antigen binding site of an antibody, e.g., all or part of theheavy and/or light chain variable domain, such that the immunobinderspecifically recognizes a target antigen. Non-limiting examples ofimmunobinders include full-length immunoglobulin molecules and scFvs, aswell as antibody fragments, including but not limited to (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H)1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fab′ fragment, which is essentially a Fab with part ofthe hinge region (see, Fundamental Immunology (Paul ed., 3.sup.rd ed.1993); (iv) a Fd fragment consisting of the V_(H) and C_(H)1 domains;(v) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (vi) a single domain antibody such as a Dab fragment(Ward et al., (1989) Nature 341:544-546), which consists of a V_(H) orV_(L) domain, a Camelid (see Hamers-Casterman, et al., Nature363:446-448 (1993), and Dumoulin, et al., Protein Science 11:500-515(2002)) or a Shark antibody (e.g., shark Ig-NARs Nanobodies®); and (vii)a nanobody, a heavy chain region containing the variable domain and twoconstant domains.

As used herein, the term “functional property” is a property of apolypeptide (e.g., an immunobinder) for which an improvement (e.g.,relative to a conventional polypeptide) is desirable and/or advantageousto one of skill in the art, e.g., in order to improve the manufacturingproperties or therapeutic efficacy of the polypeptide. In oneembodiment, the functional property is stability (e.g., thermalstability). In another embodiment, the functional property is solubility(e.g., under cellular conditions). In yet another embodiment, thefunctional property is non-aggregation. In still another embodiment, thefunctional property is protein expression (e.g., in a prokaryotic cell).In yet another embodiment the functional property is a refoldingefficiency following an inclusion body solubilization in a correspondingpurification process. In certain embodiments, antigen binding affinityis not a functional property desired for improvement.

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., on VEGF) to which an immunoglobulin or antibodyspecifically binds. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive aminoacids in a unique spatial conformation. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996).

The terms “specific binding,” “selective binding,” “selectively binds,”and “specifically binds,” refer to antibody binding to an epitope on apredetermined antigen. Typically, the antibody binds with an affinity(K_(D)) of approximately less than 10⁻⁷ M, such as approximately lessthan 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower.

The term “K_(D),” or “K_(d)” refers to the dissociation equilibriumconstant of a particular antibody-antigen interaction. Typically, theantibodies of the invention bind to VEGF with a dissociation equilibriumconstant (K_(D)) of less than approximately 10⁻⁷ M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower, for example, asdetermined using surface plasmon resonance (SPR) technology in a BIACOREinstrument.

The terms “neutralizes VEGF,” “inhibits VEGF,” and “blocks VEGF” areused interchangeably to refer to the ability of an antibody of theinvention to prevent VEGF from interacting with one or more VEGFreceptors such as VEGFR-1 and/or VEGFR-2, and, for example, triggeringsignal transduction.

A “recombinant immunobinder” as used herein refers to an immunobinderbeing produced by expression from recombinant DNA.

A “chimeric” immunobinder as used herein has a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies. Humanizedantibody as used herein is a subset of chimeric antibodies.

“Humanized antibodies” as used herein are immunobinders that have beensynthesized using recombinant DNA technology to circumvent immuneresponse to foreign antigens. Humanization is a well-establishedtechnique for reducing the immunogenicity of monoclonal antibodies ofxenogenic sources. This involves the choice of an acceptor framework,preferably a human acceptor framework, the extent of the CDRs from thedonor immunobinder to be inserted into the acceptor framework and thesubstitution of residues from the donor framework into the acceptorframework. A general method for grafting CDRs into human acceptorframeworks has been disclosed by Winter in U.S. Pat. No. 5,225,539,which is hereby incorporated by reference in its entirety. U.S. Pat. No.6,407,213 the teachings of which are incorporated by reference in itsentirety, discloses a number of amino acid positions of the frameworkwhere a substitution from the donor immunobinder is preferred.

The term “nucleic acid molecule,” refers to DNA molecules and RNAmolecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. A nucleic acidis “operably linked” when it is placed into a functional relationshipwith another nucleic acid sequence. For instance, a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence.

The term “vector,” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.

The term “host cell” refers to a cell into which an expression vectorhas been introduced. Host cells can include bacterial, microbial, plantor animal cells. Bacteria, which are susceptible to transformation,include members of the enterobacteriaceae, such as strains ofEscherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis;Pneumococcus; Streptococcus, and Haemophilus influenzae. Suitablemicrobes include Saccharomyces cerevisiae and Pichia pastoris. Suitableanimal host cell lines include CHO (Chinese Hamster Ovary lines) and NS0cells.

The terms “treat,” “treating,” and “treatment,” refer to therapeutic orpreventative measures described herein. The methods of “treatment”employ administration to a subject, in need of such treatment, anantibody of the present invention, for example, a subject having aVEGF-mediated disorder or a subject who ultimately may acquire such adisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disorder or recurring disorder,or in order to prolong the survival of a subject beyond that expected inthe absence of such treatment.

The term “VEGF-mediated disorder” refers to any disorder, the onset,progression or the persistence of the symptoms or disease states ofwhich requires the participation of VEGF. Exemplary VEGF-mediateddisorders include, but are not limited to, age-related maculardegeneration, neovascular glaucoma, diabetic retinopathy, retinopathy ofprematurity, retrolental fibroplasia, breast carcinomas, lungcarcinomas, gastric carcinomas, esophageal carcinomas, colorectalcarcinomas, liver carcinomas, ovarian carcinomas, the comas,arrhenoblastomas, cervical carcinomas, endometrial carcinoma,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas,hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma,cavernous hemangioma, hemangioblastoma, pancreas carcinomas,retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as that associated with brain tumors), Meigs' syndrome, rheumatoidarthritis, psoriasis and atherosclerosis.

The term “effective dose” or “effective dosage” refers to an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

The term “subject” refers to any human or non-human animal. For example,the methods and compositions of the present invention can be used totreat a subject with a VEGF-mediated disorder.

The term “Min-graft” or “min” as used herein refers to a humanizedvariable domain that was generated by grafting of rabbit CDRs from arabbit variable domain into a naturally occurring human acceptorframework (FW 1.4, SEQ ID No. 172). No changes in the framework regionsare made. The framework itself was preselected for desirable functionalproperties (solubility and stability).

The term “Max-graft” or “max” as used herein refers to a humanizedvariable domain that was generated by grafting of rabbit CDRs from arabbit variable domain into the “rabbitized”, human acceptor framework“RabTor” (rFW1.4, SEQ ID No. 173), or into a derivative thereof referredto as rFW1.4(v2) (SEQ ID No. 174). The “RabTor” framework was preparedby incorporating conserved rabbit residues (otherwise which are rathervariable in other species) at framework positions generally involved inrabbit variable domain structure and stability, with the aim to generatea universally applicable framework that accepts virtually any set ofrabbit CDRs without the need to graft donor framework residues otherthan at positions that are different in their presumable progenitorsequence, e.g. that were altered during somatic hypermutation and thus,possibly contribute to antigen binding. The presumable progenitorsequence is defined to be the closest rabbit germline counterpart and incase the closest germline counterpart could can not be established, therabbit subgroup consensus or the consensus of rabbit sequences with ahigh percentage of similarity.

The term “Min-Max” or “minmax” as used herein refers to a humanizedvariable domain comprising of a “Min-graft” variable light chaincombined with a “Max-graft” variable heavy chain.

The term “Max-Min” or “maxmin” as used herein refers to a humanizedvariable domain comprising of a “Max-graft” variable light chaincombined with a “Min-graft” variable heavy chain.

Different nomenclatures were used for the generated immunobinders. Theseare typically identified by a number (e.g. #578). In those cases where aprefix such as EP or Epi was used (e.g. EP 578 which is identical to Epi578), the same immunobinder is thereby indicated. Occasionally, animmunobinder received a second designation which is identified by theprefix “ESBA”. For example ESBA903 designates the same immunobinder as578minmax or EP578minmax or Epi578minmax.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending onthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

Anti-VEGF Immunobinders

In one aspect, the present invention provides immunobinders that bindVEGF and thus are suitable to block the function of VEGF in vivo. TheCDRs of these immunobinders are derived from rabbit anti-VEGF monoclonalantibodies which were obtained from rabbits that were immunized withhuman VEGF and/or a fragment thereof (SEQ ID No.1). To our knowledge,this is the first time that monoclonal anti-VEGF antibodies wereobtained from rabbits and characterized in detail. Surprisingly, theaffinities (Kd) were found to be extraordinarily high.

In certain embodiments, the invention provides an immunobinder, whichspecifically binds VEGF, comprising at least one of a CDRH1, a CDRH2, aCDRH3, a CDRL1, a CDRL2, or a CDRL3 amino acid sequence. Exemplary CDRamino acid sequences for use in the immunobinders of the invention areset forth in SEQ ID Nos: 2-72 (Tables 1-6).

TABLE 1 CDR H1 amino acid sequences of anti-VEGFimmunobinders of the invention. Sequence Identifier CDR-H1 SEQ ID No.60-11-4 GFPFSSGYWVC 2 60-11-6 GFSFSSGYWIC 3 435 GFSLNTNYWMC 4 453GFSFSRSYYIY 5 375 GFSFTTTDYMC 6 610 GIDFSGAYYMG 7 578 GFSLTDYYYMT 8 534GFSLSYYYMS 9 567 GFSLSDYYMC 10 509 GFSLSSYYMC 11 511 GFSLNTYYMN 12509maxll GFSLSSYYMS 13 Consensus GFSLSSGYYMC 14

TABLE 2 CDR H2 amino acid sequences of antiVEGFimmunobinders of the invention. Sequence Identifier CDR-H2 SEQ ID No. 60 CIYAGSSGSTYYASWAKG 15 435 CMYTGSYNRAYYASWAKG 16 453CIDAGSSGILVYANWAKG 17 375 CILAGDGSTYYANWAKG 18 610 YIDYDGDRYYASWAKG 19578 FIDPDDDPYYATWAKG 20 534 IIGPGDYTDYASWAKG 21 567 CLDYFGSTDDASWAKG 22509 CLDYVGDTDYASWAKG 23 511 IIAPDDTTYYASWAKS 24 509maxllILDYVGDTDYASWAKG 25 Consensus CIDAGSDGDTYYASWAKG 26

TABLE 3 CDR H3 amino acid sequences of antiVEGFimmunobinders of the invention. Sequence Identifier CDR-H3 SEQ ID No. 60 GNNYYIYTDGGYAYAGLEL 27 435 GSNWYSDL 28 453 GDASYGVDSFMLPL 29 375SDPASSWSFAL 30 610 SDYSSGWGTDI 31 578 GDHNSGWGLDI 32 534 GDDNSGWGEDI 33567 TDDSRGWGLNI 34 509 TDDSRGWGLNI 35 511 SGDTTAWGADI 36 ConsensusGDDSSGYTDGGYAYWGLDI 37

TABLE 4 CDR Ll amino acid sequences of anti-VEGFimmunobinders of the invention. Sequence Identifier CDR-L1 SEQ ID No. 60 QASQSISSYLS 38 435 QASQSIGSSLA 39 453 QSSQSVWNNNRLA 40 375QASENINIWLS 41 610 QASQSISSWLS 42 578 QASEIIHSWLA 43 534 QASQSINIWLS 44567 QADQSIYIWLS 45 509 QASQNIRIWLS 46 511 QASQSINIWCS 47 511maxQASQSINIWLS 48 Consensus QASQSININNWLS 49

TABLE 5 CDR L2 amino acid sequences of anti-VEGFimmunobinders of the invention. Sequence Identifier CDR-L2 SEQ ID No. 60 KASTLAS 50 435 TAANLAS 51 453 YASTLAS 52 375 QASKLAS 53 610 QASTLAS54 578 LASTLAS 55 534 KESTLAS 56 567 KASTLES 57 509 KASTLES 58 511RASTLAS 59 Consensus KASTLAS 60

TABLE 6 CDR L3 amino acid sequences of anti-VEGFimmunobinders of the invention. Sequence Identifier CDR-L3 SEQ ID No. 60 QSNYGGSSSDYGNP 61 435 QNFATSDTVT 62 453 AGGYSSTSDNT 63 375QNNYSYNRYGAP 64 610 QNNYGFRSYGGA 65 578 QNVYLASTNGAN 66 534 QNNYDSGNNGFP67 567 QNNAHYSTNGGT 68 509 QNNAHYSTNGGT 69 511 QANYAYSAGYGAA 70Consensus QNNYHYSSSTNGGT 71

In one embodiment, the invention provides an immunobinder comprising atleast one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a consensus sequence of the group consistingof SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 37, SEQ ID NO: 49, SEQ IDNO: 60 and SEQ ID NO: 71. Preferably, the VH of said immunobindercomprise the CDRs of the group consisting of SEQ ID NO: 14, SEQ ID NO:26 and SEQ ID NO: 37 and/or the CDRs of the VL of said immunobindercomprise CDRs of the group consisting of SEQ ID NO: 49, SEQ ID NO: 60and SEQ ID NO: 71. Preferably, the CDR is selected from the groupconsisting of SEQ ID NO: 2 to SEQ ID NO: 13, SEQ ID NO: 15 to SEQ ID NO:25, SEQ ID NO: 27 to SEQ ID NO: 36, SEQ ID NO: 38 to SEQ ID NO: 48, SEQID NO: 50 to SEQ ID NO: 59 and SEQ ID NO: 61 to SEQ ID NO: 70.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 38, SEQ IDNO: 50 and SEQ ID NO: 61. Preferably, the VH of said immunobindercomprise the CDRs of the group consisting of SEQ ID NO: 2, SEQ ID NO: 15and SEQ ID NO: 27 and/or the CDRs of the VL of said immunobindercomprise CDRs of the group consisting of SEQ ID NO: 38, SEQ ID NO: 50and SEQ ID NO: 61. In another preferred embodiment, the VH of saidimmunobinder comprise the CDRs of the group consisting of SEQ ID NO: 3,SEQ ID NO: 15 and SEQ ID NO: 27 and/or the CDRs of the VL of saidimmunobinder comprise CDRs of the group consisting of SEQ ID NO: 38, SEQID NO: 50 and SEQ ID NO: 61.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 4, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 51, andSEQ ID NO: 62. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 28and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 39, SEQ ID NO: 51, and SEQ ID NO: 62.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 5, SEQ ID NO: 17, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 52 andSEQ ID NO: 63. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 29and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 40, SEQ ID NO: 52 and SEQ ID NO: 63.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 6, SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 41, SEQ ID NO: 53 andSEQ ID NO: 64. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 6, SEQ ID NO: 18 and SEQ ID NO: 30and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 41, SEQ ID NO: 53 and SEQ ID NO: 64.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 7, SEQ ID NO: 19, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 54 andSEQ ID NO: 65. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 7, SEQ ID NO: 19 and SEQ ID NO: 31and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 42, SEQ ID NO: 54 and SEQ ID NO: 65.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 8, SEQ ID NO: 20, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 55 andSEQ ID NO: 66. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 32and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 43, SEQ ID NO: 55 and SEQ ID NO: 66.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 9, SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 56 andSEQ ID NO: 67. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 9, SEQ ID NO: 21 and SEQ ID NO: 33and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 44, SEQ ID NO: 56 and SEQ ID NO: 67.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 10, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 57 andSEQ ID NO: 68. Preferably, the VH of said immunobinder comprise the CDRsof the group consisting of SEQ ID NO: 10, SEQ ID NO: 22 and SEQ ID NO:34 and/or the CDRs of the VL of said immunobinder comprise CDRs of thegroup consisting of SEQ ID NO: 45, SEQ ID NO: 57 and SEQ ID NO: 68

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 35, SEQID NO: 46, SEQ ID NO: 58 and SEQ ID NO: 69. Preferably, the VH of saidimmunobinder comprise the CDRs of the group consisting of SEQ ID NO: 11,SEQ ID NO: 23 and SEQ ID NO: 35 and/or the CDRs of the VL of saidimmunobinder comprise CDRs of the group consisting of SEQ ID NO: 46, SEQID NO: 58 and SEQ ID NO: 69. Alternatively, the VH of said immunobindercomprise the CDRs of the group consisting of SEQ ID NO: 13, SEQ ID NO:25 and SEQ ID NO: 35 and/or the CDRs of the VL of said immunobindercomprise CDRs of the group consisting of SEQ ID NO: 46, SEQ ID NO: 58and SEQ ID NO: 69.

In another embodiment, the invention provides an immunobinder comprisingat least one CDR having at least 75% similarity, preferably at least 75%identity, more preferably at least 80%, 85%, 90% 95%, even morepreferably 100% identity to a sequence of the group consisting of SEQ IDNO: 12, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 59, and SEQ ID NO: 70. Preferably, the VH of said immunobindercomprise the CDRs of the group consisting of SEQ ID NO: 12, SEQ ID NO:24 and SEQ ID NO: 36. Additionally or alternatively, the VL of saidimmunobinder comprise CDRs of the group consisting of SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 59, and SEQ ID NO: 70, e.g. SEQ ID NO: 47, SEQ IDNO: 59, and SEQ ID NO: 70; or SEQ ID NO: 48, SEQ ID NO: 59, and SEQ IDNO: 70.

In a much preferred embodiment, the immunobinder disclosed hereinneutralizes human VEGF and is cross-reactive with rat/mouse VEGF or aportion thereof.

The immunobinder can comprise an antibody or any alternative bindingscaffold capable of accommodating CDRs. The CDRs set forth in SEQ IDNos: 2-72 can be grafted onto any suitable binding scaffold using anyart recognized methods (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.). However, it is preferred that theimmunobinders disclosed herein are humanized, and thus suitable fortherapeutic applications.

In the case of antibodies, the rabbit CDRs set forth in SEQ ID Nos: 2-72may be grafted into the framework regions of any antibody from anyspecies. However, it has previously been discovered that antibodies orantibody derivatives comprising the frameworks identified in the socalled “quality control” screen (WO0148017) are characterised by agenerally high stability and/or solubility and thus may also be usefulin the context of extracellular applications such as neutralizing humanVEGF. Moreover, it has further been discovered that one particularcombination of these VL (variable light chain) and VH (variable heavychain) soluble and stable frameworks is particularly suited toaccommodating rabbit CDRs. Accordingly, in one embodiment, the CDRs setforth in SEQ ID Nos: 2-72 are grafted into the human antibody frameworksderived by “quality control” screening disclosed in EP1479694. The aminoacid sequences of exemplary frameworks for use in the invention are setforth in SEQ ID Nos: 172 to 174. It was surprisingly found that upongrafting into said framework or its derivatives, loop conformation of alarge variety of rabbit CDRs could be fully maintained, largelyindependent of the sequence of the donor framework. Moreover, saidframework or its derivatves containing different rabbit CDRs are wellexpressed and produced contrary to the rabbit wild type single chainsand still almost fully retain the affinity of the original donor rabbitantibodies.

Thus, in a preferred embodiment, the CDRs and/or CDR motifs disclosedherein are present in a heavy chain variable region framework sequencehaving at least 80% sequence identity, more preferably at least 85%, 90%95%, even more preferably 100% identity to the sequence of SEQ ID NO:169. In a preferred embodiment, the heavy chain variable regionframework sequence comprises SEQ ID NO: 170 or SEQ ID NO: 171.

In a preferred embodiment, the CDRs and/or CDR motifs disclosed hereinare present in a light chain variable region framework sequence havingat least 85% sequence identity, more preferably at least 90%, 95%, evenmore preferably 100% identity to the sequence of SEQ ID NO: 167, morepreferably comprising SEQ ID NO: 167 or SEQ ID NO: 168.

In rabbit antibodies, CDRs can contain cysteine residues that becomedisulphide linked to cysteine residues in the antibody framework.Accordingly, it may be necessary, when grafting rabbit CDRs containingcysteine residues into non-rabbit framework regions to introducecysteine residues in the non-rabbit framework by, for example,mutagenesis to facilitate the stabilization of rabbit CDR through adisulphide linkage.

In other embodiments, the invention provides an immunobinder, whichspecifically binds VEGF, comprising at least one of a VL or a VH aminoacid sequence. Exemplary VH or VL amino acid sequences for use in theimmunobinders of the invention are set forth in SEQ ID Nos: 72-106 and107-166, respectively.

In a preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 118, SEQ ID NO:119, SEQ ID NO: 130 and SEQ ID NO: 131 (VH 60-11-4, VH 60-11-6, VH60-11-4 min, VH 60-11-6 min, VH 60-11-4-max and VH 60-11-6max,respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 72, SEQ ID NO:82 and SEQ ID NO: 93 (VL 60, VL60 min, VL 60max, respectively).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 109, SEQ ID NO: 120 and SEQ ID NO: 132 (VH 435,VH 435 min and VH 435max, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 73, SEQ ID NO: 83 and SEQ ID NO:94 (VL 435, VL435 min and VL 435max, respectively).

Preferably, said immunobinder has at least 80%, more preferably at least85%, 90%, 95%, most preferably 100% identity to SEQ ID NO: 175 (435max).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 110, SEQ ID NO: 121 and SEQ ID NO: 133 (VH 453,VH 453 min and VH 453max, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 74, SEQ ID NO:84 and SEQ ID NO: 95(VL 453, VL453 min and VL 453max, respectively).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 111, SEQ ID NO: 122 and SEQ ID NO: 134 (VH 375,VH 375 min and VH 375max, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 75, SEQ ID NO: 85 and SEQ ID NO:96 (VL 375, VL375 min and VL 375max, respectively).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 112, SEQ ID NO: 123 and 135 (VH 610, VH 610 minand VH 610max, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 76, SEQ ID NO: 86 and SEQ ID NO: 97 (VL 610, VL610 min and VL 610max, respectively).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 113, SEQ ID NO: 124, SEQ ID NO: 129, SEQ ID NO:136, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 147, SEQID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO:152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO: 155, SEQ ID NO: 156, SEQID NO:157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO:161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165 andSEQ ID NO: 166 (VH 578 and variants thereof);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 92, SEQ ID NO:98, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105 (VL 578 andvariants thereof).

Preferably, said immunobinder has at least 80%, more preferably at least85%, 90%, 95%, most preferably 100% identity to SEQ ID NO: 178 (578min), SEQ ID NO: 179 (578max) or SEQ ID NO: 180 (578minmax).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 114, SEQ ID NO: 125 and SEQ ID NO: 137 (VH 534,VH 534 min and VH 534max, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 78, SEQ ID NO: 88 and SEQ ID NO: 99 (VL 534, VL534 min and VL 534max, respectively).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO:138 and SEQ IDNO: 143 (VH 567, VH 567 min and VH 567max, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO:. 79, SEQ ID NO:89 and SEQ ID NO: 100 (VL 567,VL 567 min and VL 567max, respectively).

Preferably, said immunobinder has at least 80%, more preferably at least85%, 90%, 95%, most preferably 100% identity to SEQ ID NO: 177 (567min).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 116, SEQ ID NO: 127, SEQ ID NO:139 and SEQ IDNO: 140 (VH 509, VH 509 min, VH 509max and VH 509maxII, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 80, SEQ ID NO: 90 and SEQ ID NO: 101 (VL 509,VL 509 min and VL 509max, respectively).

In another preferred embodiment, the invention provides an immunobindercomprising a VH having at least 80%, more preferably at least 85%, 90%,95%, most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 117, SEQ ID NO: 128, SEQ ID NO: 141 and SEQ IDNO: 145 (VH 511, VH 511 min, VH 511max and VH 511maxDHP, respectively);

and/or a VL having at least 80%, more preferably at least 85%, 90%, 95%,most preferably 100% identity to a sequence selected from the groupconsisting of SEQ ID NO: 81, SEQ ID NO: 91, SEQ ID NO: 102 and SEQ IDNO: 106 (VL 511, VL 511 min, VL 511max and VL 511minC41L, respectively).

Preferably, said immunobinder has at least 80%, more preferably at least85%, 90%, 95%, most preferably 100% identity to SEQ ID NO: 176(511_max).

In certain embodiments, the invention further provides an immunobinder,which specifically binds VEGF, comprising an amino acid sequence withsubstantial similarity to an amino acid sequence set forth in SEQ IDNos: 2-166 and in SEQ ID Nos: 175-180, and wherein the immunobinderessentially retains or improves the desired functional properties of theanti-VEGF immunobinder of the invention. Preferred percentagesimilarities include, but are not limited to, at least 50%, 60%, 70%,75%, 80%, 85%, 90% or 95% identity.

In certain embodiments, the invention further provides an immunobinder,which specifically binds VEGF, comprising an amino acid sequence withsubstantial identity to an amino acid sequence set forth in SEQ ID Nos:2-166 and in SEQ ID Nos. 175-180, and wherein the immunobinder retainsor improves the desired functional properties of the anti-VEGFimmunobinder of the invention. Preferred percentage identities include,but are not limited to, at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or95% identity.

In certain embodiments, the invention further provides an immunobinder,which specifically binds VEGF, comprising an amino acid sequence withconservative substitutions relative to an amino acid sequence set forthin SEQ ID Nos: 2-166 and in SEQ ID Nos. 175-180, and wherein theimmunobinder retains or improves the desired functional properties ofthe anti-VEGF immunobinder of the invention.

In some embodiments, the invention provides immunobinders that bindspecifically to human VEGF and cross react with VEGF molecules of otherspecies, for example, mouse VEGF, rat VEGF, rabbit VEGF or guinea pigVEGF. In a particular embodiment the anti-VEGF immunobinder can bindspecifically to human and rat/mouse VEGF.

In some embodiments, the invention provides immunobinders that bindspecifically to human VEGF and do not cross react with VEGF molecules ofother species, for example, mouse VEGF, rat VEGF, rabbit VEGF or guineapig VEGF.

In some embodiments, the invention provides immunobinders that bindspecifically to human VEGF and wherein the immunobinders are affinitymatured.

In one embodiment, antibodies and antibody fragments of the presentinvention are single chain antibodies (scFv) or Fab fragments. In thecase of scFv antibodies, a selected VL domain can be linked to aselected VH domain in either orientation by a flexible linker. Asuitable state of the art linker consists of repeated GGGGS amino acidsequences or variants thereof. In a preferred embodiment of the presentinvention a (GGGGS)₄ linker of the amino acid sequence set forth in SEQID NO: 181, but variants of 1-3 repeats are also possible (Holliger etal. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers thatcan be used for the present invention are described by Alfthan et al.(1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al.(1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), CancerImmunol. Immunother. 50:51-59. The arrangement can be eitherVL-linker-VH or VH-linker-VL, with the former orientation being thepreferred one. However, single VH or VL domain antibodies are alsocontemplated. In the case of Fab fragments, selected light chainvariable domains VL are fused to the constant region of a human Ig kappachain, while the suitable heavy chain variable domains VH are fused tothe first (N-terminal) constant domain CH1 of a human IgG. At theC-terminus of the constant domain or at other sites of the variable orconstant domain, an inter-chain disulfide bridge may be formed.Alternatively, the two chains may also be linked by a flexible linkerresulting in a single chain Fab antibody.

The antibodies or antibody derivatives of the present invention can haveaffinities to human VEGF with dissociation constants K_(d) in a range of10⁻¹⁴M to 10⁻⁵M. In a preferred embodiment of the present invention theK_(d) is ≦1 nM. The affinity of an antibody for an antigen can bedetermined experimentally using a suitable method (Berzofsky et al.“Antibody-Antigen Interactions”, in Fundamental Immunology, Paul, W. E.,Ed, Raven Press: New York, N.Y. (1992); Kuby, J. Immunology, W.H.Freeman and Company: New York, N.Y.) and methods described therein.

The company Epitomics sells an anti-VEGF antibody which is a rabbitmonoclonal antibody (VEGF (C-term) Rabbit Antibody, Cat. no. 1909-1).Said antibody is directed against residues on the C-terminus of humanVEGF and therefore not able to neutralize VEGF. Hence, said antibody isnot suitable for therapeutic applications. Moreover, said monoclonal IgGis not a humanized antibody but is a natural rabbit full-lengthimmunoglobulin. In addition, it was shown that this antibody does notrecognize the native form of VEGF.

Immunobinders that Bind the Same Epitopes on VEGF

In another aspect, the invention provides antibodies that bind to anepitope on VEGF recognized by an antibody comprising any one of theamino acid sequences set forth in SEQ ID No 2-211. Such antibodies canbe identified based upon their ability to cross-compete with an antibodycomprising any one or more of the amino acid sequences set forth in SEQID No 2-211 in standard VEGF-binding assays including, but not limited,to ELISA. The ability of a test antibody to inhibit the binding to humanVEGF of an antibody comprising any one or more of the amino acidsequences set forth in SEQ ID No 2-211 demonstrates that the testantibody can cross-compete thus interact with an overlapping epitope onhuman VEGF as an antibody comprising any one or more of the amino acidsequences set forth in SEQ ID No 2-211.

Additionaly or alternatively, such antibodies can be also identifiedusing standard epitope mapping techniques to determine if they bind tothe same peptide immunogens. Structural modelling techniques may also beemployed to further define the precise molecular determinants to theantibody/VEGF interaction, including, but not limited to, NMR, X-raycrystallography, computer based modeling, or protein tomography (Banyayet al, 2004 ASSAY and Drug Development Technologies (2), 5, Page516-567). Indeed, the crystal structure of VEGF has been solved and thesurface amino acid residues involved in VEGFr binding are known (Fuh, etal., 2006, J. Biol. Chem., 281, 6625-6631). Accordingly, given the aminoacid sequence of the peptide immunogen and the structural knowledge ofVEGF available in the art, it is well within the skill in art toidentify antibodies that bind to an epitope on VEGF recognized by theantibodies comprising any one or more of the amino acid sequences setforth in SEQ ID No 2-211.

In some embodiments, antibodies that bind to an epitope on VEGFrecognized by an antibody comprising any one or more of the amino acidsequences set forth in SEQ ID No 2-211 bind to VEGF with an affinity ofat least 10⁷ M⁻¹, for example, at least 10⁷ M⁻¹, at least 10⁸ M⁻¹, atleast 10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 10¹² M⁻¹or at least 10¹³ M⁻¹.

In some embodiments, antibodies that bind to an epitope on VEGFrecognized by an antibody comprising any one one or more of the aminoacid sequences set forth in SEQ ID No 2-211 bind specifically to humanVEGF and do not cross react with VEGF molecules of other species, forexample, mouse VEGF, rat VGEF, rabbit VEGF, or guinea pig VEGF.

In some embodiments, antibodies that bind to an epitope on VEGFrecognized by an antibody comprising any one or more of the amino acidsequences set forth in SEQ ID No 2-211 cross react with VEGF moleculesof other species, for example, mouse VEGF, rat VEGF, or rabbit VEGF.

Optimized Variants

The antibodies of the invention may be further optimized for enhancedfunctional properties, e.g., for enhanced solubility and/or stability.

In certain embodiments, the antibodies of the invention are optimizedaccording to the “functional consensus” methodology disclosed in PCTApplication Serial No. PCT/EP2008/001958, entitled “Sequence BasedEngineering and Optimization of Single Chain Antibodies”, filed on Mar.12, 2008, which is incorporated herein by reference. For example, theVEGF immunobinders of the invention can be compared with a database offunctionally-selected scFvs to identify amino acid residue positionsthat are either more or less tolerant of variability than thecorresponding position(s) in the VEGF immunobinder, thereby indicatingthat such identified residue position(s) may be suitable for engineeringto improve functionality such as stability and/or solubility.

Exemplary framework positions for substitution are described in PCTApplication No. PCT/CH2008/000285, entitled “Methods of ModifyingAntibodies, and Modified Antibodies with Improved FunctionalProperties”, filed on Jun. 25, 2008, and PCT Application No.PCT/CH2008/000284, entitled “Sequence Based Engineering and Optimizationof Single Chain Antibodies”, filed on Jun. 25, 2008. For example, one ormore of the following substitutions may be introduced at an amino acidposition (AHo numbering is referenced for each of the amino acidposition listed below) in the heavy chain variable region of animmunobinder of the invention:

-   -   (a) Q or E at amino acid position 1;    -   (b) Q or E at amino acid position 6;    -   (c) T, S or A at amino acid position 7, more preferably T or A,        even more preferably T;    -   (d) A, T, P, V or D, more preferably T, P, V or D, at amino acid        position 10,    -   (e) L or V, more preferably L, at amino acid position 12,    -   (f) V, R, Q, M or K, more preferably V, R, Q or M at amino acid        position 13;    -   (g) R, M, E, Q or K, more preferably R, M, E or Q, even more        preferably R or E, at amino acid position 14;    -   (h) L or V, more preferably L, at amino acid position 19;    -   (i) R, T, K or N, more preferably R, T or N, even more        preferably N, at amino acid position 20;    -   (j) I, F, L or V, more preferably I, F or L, even more        preferably I or L, at amino acid position 21;    -   (k) R or K, more preferably K, at amino acid position 45;    -   (l) T, P, V, A or R, more preferably T, P, V or R, even more        preferably R, at amino acid position 47;    -   (m) K, Q, H or E, more preferably K, H or E, even more        preferably K, at amino acid position 50;    -   (n) M or I, more preferably I, at amino acid position 55;    -   (o) K or R, more preferably K, at amino acid position 77;    -   (p) A, V, L or I, more preferably A, L or I, even more        preferably A, at amino acid position 78;    -   (q) E, R, T or A, more preferably E, T or A, even more        preferably E, at amino acid position 82;    -   (r) T, S, I or L, more preferably T, S or L, even more        preferably T, at amino acid position 86;    -   (s) D, S, N or G, more preferably D, N or G, even more        preferably N, at amino acid position 87;    -   (t) A, V, L or F, more preferably A, V or F, even more        preferably V, at amino acid position 89;    -   (u) F, S, H, D or Y, more preferably F, S, H or D, at amino acid        position 90;    -   (v) D, Q or E, more preferably D or Q, even more preferably D,        at amino acid position 92;    -   (w) G, N, T or S, more preferably G, N or T, even more        preferably G, at amino acid position 95;    -   (x) T, A, P, F or S, more preferably T, A, P or F, even more        preferably F, at amino acid position 98;    -   (y) R, Q, V, I, M, F, or L, more preferably R, Q, I, M, F or L,        even more preferably Y, even more preferably L, at amino acid        position 103; and    -   (z) N, S or A, more preferably N or S, even more preferably N,        at amino acid position 107.

Additionally or alternatively, one or more of the followingsubstitutions can be introduced into the light chain variable region ofan immunobinder of the invention:

-   -   (aa) Q, D, L, E, S, or I, more preferably L, E, S or I, even        more preferably L or E, at amino acid position 1;    -   (bb) S, A, Y, I, P or T, more preferably A, Y, I, P or T, even        more preferably P or T at amino acid position 2;    -   (cc) Q, V, T or I, more preferably V, T or I, even more        preferably V or T, at amino acid position 3;    -   (dd) V, L, I or M, more preferably V or L, at amino acid        position 4;    -   (ee) S, E or P, more preferably S or E, even more preferably S,        at amino acid position 7;    -   (ff) T or I, more preferably I, at amino acid position 10;    -   (gg) A or V, more preferably A, at amino acid position 11;    -   (hh) S or Y, more preferably Y, at amino acid position 12;    -   (ii) T, S or A, more preferably T or S, even more preferably T,        at amino acid position 14;    -   (jj) S or R, more preferably S, at amino acid position 18;    -   (kk) T or R, more preferably R, at amino acid position 20;    -   (ll) R or Q, more preferably Q, at amino acid position 24;    -   (mm) H or Q, more preferably H, at amino acid position 46;    -   (nn) K, R or I, more preferably R or I, even more preferably R,        at amino acid position 47;    -   (oo) R, Q, K, E, T, or M, more preferably Q, K, E, T or M, at        amino acid position 50;    -   (pp) K, T, S, N, Q or P, more preferably T, S, N, Q or P, at        amino acid position 53;    -   (qq) I or M, more preferably M, at amino acid position 56;    -   (rr) H, S, F or Y, more preferably H, S or F, at amino acid        position 57;    -   (ss) I, V or T, more preferably V or T, R, even more preferably        T, at amino acid position 74;    -   (tt) R, Q or K, more preferably R or Q, even more preferably R,        at amino acid position 82;    -   (uu) L or F, more preferably F, at amino acid position 91;    -   (vv) G, D, T or A, more preferably G, D or T, even more        preferably T, at amino acid position 92;    -   (xx) S or N, more preferably N, at amino acid position 94;    -   (yy) F, Y or S, more preferably Y or S, even more preferably S,        at amino acid position 101; and    -   (zz) D, F, H, E, L, A, T, V, S, G or I, more preferably H, E, L,        A, T, V, S, G or I, even more preferably A or V, at amino acid        position 103.

The AHo numbering system is described further in Honegger, A. andPluckthun, A. (2001) J. Mol. Biol. 309:657-670). Alternatively, theKabat numbering system as described further in Kabat et al. (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) may be used. Conversion tables for the two differentnumbering systems used to identify amino acid residue positions inantibody heavy and light chain variable regions are provided in A.Honegger, J. Mol. Biol. 309 (2001) 657-670.

In other embodiments, the immunobinders of the invention comprise one ormore of the solubility and/or stability enhancing mutations described inU.S. Provisional Application Ser. No. 61/075,692, entitled “SolubilityOptimization of Immunobinders,” filed on Jun. 25, 2008. In certainpreferred embodiments, the immunobinder comprises a solubility enhancingmutation at an amino acid position selected from the group of heavychain amino acid positions consisting of 12, 103 and 144 (AHo Numberingconvention). In one preferred embodiment, the immunobinder comprises oneor more substitutions selected from the group consisting of: (a) Serine(S) at heavy chain amino acid position 12; (b) Serine (S) or Threonine(T) at heavy chain amino acid position 103; and (c) Serine (s) orThreonine (T) at heavy chain amino acid position 144. In anotherembodiment, the immunobinder comprises the following substitutions: (a)Serine (S) at heavy chain amino acid position 12; (b) Serine (S) orThreonine (T) at heavy chain amino acid position 103; and (c) Serine (S)or Threonine (T) at heavy chain amino acid position 144.

Hybridomas Expressing Rabbit Anti-VEGF Antibodies

In another aspect, the invention provides a hybridoma expressing amonoclonal antibody comprising any one or more of the amino acidsequences set forth in SEQ ID Nos 72-81. Methods for generatinghybridomas from Rabbit B-cells are well known in the art and aredisclosed, for example, in U.S. patent application 2005/0033031.

Production of Anti-VEGF Immunobinders

The antibodies or antibody derivatives of the present invention may begenerated using routine techniques in the field of recombinant genetics.Knowing the sequences of the polypeptides, the cDNAs encoding them canbe generated by gene synthesis (www.genscript.com). These cDNAs can becloned into suitable vector plasmids. Once the DNA encoding a VL and/ora VH domain are obtained, site directed mutagenesis, for example by PCRusing mutagenic primers, can be performed to obtain various derivatives.The best “starting” sequence can be chosen depending on the number ofalterations desired in the VL and/or VH sequences.

Methods for incorporating or grafting CDRs into framework regionsinclude those set forth in, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al, as well as those disclosed in U.S.Provisional Application Ser. No. 61/075,697, entitled “Humanization ofRabbit Antibodies Using Universal Antibody Frameworks,” filed on Jun.25, 2008.

Standard cloning and mutagenesis techniques well known to the personskilled in the art can be used to attach linkers, shuffle domains orconstruct fusions for the production of Fab fragments. Basic protocolsdisclosing the general methods of this invention are described inMolecular Cloning, A Laboratory Manual (Sambrook & Russell, 3^(rd) ed.2001) and in Current Protocols in Molecular Biology (Ausubel et al.,1999).

The DNA sequence harboring a gene encoding a scFv polypeptide, or in thecase of Fab fragments, encoding either two separate genes or abi-cistronic operon comprising the two genes for the VL-Cκ and theVH-CH1 fusions are cloned in a suitable expression vector, preferablyone with an inducible promoter. Care must be taken that in front of eachgene an appropriate ribosome binding site is present that ensurestranslation. It is to be understood that the antibodies of the presentinvention comprise the disclosed sequences rather than they consist ofthem. For example, cloning strategies may require that a construct ismade from which an antibody with one or a few additional residues at theN-terminal end are present. Specifically, the methionine derived fromthe start codon may be present in the final protein in cases where ithas not been cleaved posttranslationally. Most of the constructs forscFv antibodies give rise to an additional alanine at the N-terminalend. In a preferred embodiment of the present invention, an expressionvector for periplasmic expression in E. coli is chosen (Krebber, 1997).Said vector comprises a promoter in front of a cleavable signalsequence. The coding sequence for the antibody peptide is then fused inframe to the cleavable signal sequence. This allows the targeting of theexpressed polypeptide to the bacterial periplasm where the signalsequence is cleaved. The antibody is then folded. In the case of the Fabfragments, both the VL-Cκ and the VH-CH1 fusions peptides must be linkedto an export signal. The covalent S—S bond is formed at the C-terminalcysteines after the peptides have reached the periplasm. If cytoplasmicexpression of antibodies is preferred, said antibodies usually can beobtained at high yields from inclusion bodies, which can be easilyseparated from other cellular fragments and protein. In this case theinclusion bodies are solubilized in a denaturing agent such as, e.g.,guanidine hydrochloride (GndHCl) and then refolded by renaturationprocedures well known to those skilled in the art.

Plasmids expressing the scFv or Fab polypeptides are introduced into asuitable host, preferably a bacterial, yeast or mammalian cell, mostpreferably a suitable E. coli strain as for example JM83 for periplasmicexpression or BL21 for expression in inclusion bodies. The polypeptidecan be harvested either from the periplasm or form inclusion bodies andpurified using standard techniques such as ion exchange chromatography,reversed phase chromatography, affinity chromatography and/or gelfiltration known to the person skilled in the art.

The antibodies or antibody derivatives of the present invention can becharacterized with respect to yield, solubility and stability in vitro.Binding capacities towards VEGF, preferably towards human VEGF, can betested in vitro by ELISA or surface plasmon resonance (BIACore), usingrecombinant human VEGF as described in WO9729131, the latter method alsoallowing to determine the k_(off) rate constant, which should preferablybe less than 10⁻³ s⁻¹. K_(d) values of ≦10 nM are preferred.

Aside from antibodies with strong binding affinity for human VEGF, it isalso desirable to select anti-VEGF antibodies which have otherbeneficial properties from a therapeutic perspective. For example, theantibody may be one which inhibits HUVEC cell growth in response to VEGF(see Example 3). In one embodiment, the antibody may be able to inhibitHUVEC cell proliferation in response to a near maximally effectiveconcentration of VEGF (0.08 nM). Preferably, the antibody has aneffective dose 50 (ED50) value of no more than about 5 nM, preferably nomore than about 1 nM, preferably no more than about 1 nM, preferably nomore than about 0.5 nM and most preferably no more than about 0.06 nM,for inhibiting VEGF-induced proliferation of endothelial cells in this“endothelial cell growth assay”, i.e., at these concentrations theantibody is able to inhibit VEGF-induced endothelial cell growth invitro by, e.g., 50% or more.

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-VEGF antibody, or a fragment thereof, of theinvention. An antibody of the invention, or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of theinvention may be derivatized or linked to more than one other functionalmolecule to generate multispecific molecules that bind to more than twodifferent binding sites and/or target molecules; such multispecificmolecules are also intended to be encompassed by the term “bispecificmolecule” as used herein. To create a bispecific molecule of theinvention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, tumor specific or pathogen specificantigens, peptide or binding mimetic, such that a bispecific moleculeresults. Accordingly, the present invention includes bispecificmolecules comprising at least one first binding molecule havingspecificity for VEGF and a second binding molecule having specificityfor one or more additional target epitope.

In one embodiment, the bispecific molecules of the invention comprise abinding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the invention are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be prepared byconjugating the constituent binding specificities using methods known inthe art. For example, each binding specificity of the bispecificmolecule can be generated separately and then conjugated to one another.When the binding specificities are proteins or peptides, a variety ofcoupling or cross-linking agents can be used for covalent conjugation.Examples of cross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding, for example, via the C-terminus hinge regions ofthe two heavy chains or other sites, whether naturally occurring orintroduced artificially. In a particularly preferred embodiment, thehinge region is modified to contain an odd number of sulfhydrylresidues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Further, a bispecficmolecule may be a scFv that specifically binds to first target, whereinthe VH and VL of said scFv are linked with a flexible linker comprisinga domain providing specific binding to a second target. Suitable linkersare described in U.S. Provisional Application No. 60/937,820. Methodsfor preparing bispecific molecules are described for example in U.S.Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175;U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No.5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S.Pat. No. 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or by immunoblot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the VEGF-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-VEGF complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a y counter or a scintillationcounter or by autoradiography.

Immunoconjugates

In another aspect, the present invention features an anti-VEGF antibody,or a fragment thereof, conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Suchconjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of the invention include duocarmycins,calicheamicins, maytansines and auristatins, and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst).

Cytotoxins can be conjugated to antibodies of the invention using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present invention also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for being used diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹,yttrium⁹⁰ and lutetium¹⁷⁷. Methods for preparing radioimmunconjugatesare established in the art. Examples of radioimmunoconjugates arecommercially available, including Zevalin™ (IDEC Pharmaceuticals) andBexxar™ (Corixa Pharmaceuticals), and similar methods can be used toprepare radioimmunoconjugates using the antibodies of the invention.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Uses of Anti-VEGF Antibodies

For therapeutic applications, the anti-VEGF antibodies of the inventionare administered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form such as those discussed herein, including thosethat may be administered to a human intravenously, as a bolus or bycontinuous infusion over a period of time, by topical, intraocular,intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, or inhalation routes.The antibodies also are suitably administered by intra tumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. The intraperitoneal route isexpected to be particularly useful, for example, in the treatment ofovarian tumors.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

The anti-VEGF antibodies are useful in the treatment of VEGF-mediateddiseases as described herein. For example, age-related maculardegeneration (AMD) is a leading cause of severe visual loss in theelderly population. The exudative form of AMD is characterized bychoroidal neovascularization and retinal pigment epithelial celldetachment. Because choroidal neovascularization is associated with adramatic worsening in prognosis, the VEGF antibodies of the presentinvention are especially useful in reducing the severity of AMD. Theprogress of this therapy is easily monitored by conventional techniquesincluding opthalmoscopy, ocular fundus microscopy, and ocular computertomography.

All FDA approved doses and regimes suitable for use with Lucentis areconsidered. Other doses and regimes are described in U.S. ProvisionalApplication Ser. No. 61/075,641, entitled “Improved ImmunobinderFormulations And Methods For Administration”, filed Jun. 25, 2008, andU.S. Provisional Application No. 61/058,504, which are expresslyincorporated herein.

According to another embodiment of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for those purposes, such as tumor necrosis factor(TNF), an antibody capable of inhibiting or neutralizing the angiogenicactivity of acidic or basic fibroblast growth factor (FGF) or hepatocytegrowth factor (HGF), an antibody capable of inhibiting or neutralizingthe coagulant activities of tissue factor, protein C, or protein S (seeEsmon et al., PCT Patent Publication No. WO 91/01753, published 21 Feb.1991), an antibody capable of binding to HER2 receptor (see Hudziak etal., PCT Patent Publication No. WO 89/06692, published 27 Jul. 1989), orone or more conventional therapeutic agents such as, for example,alkylating agents, photocoagulants (such as verteporfin), folic acidantagonists, anti-metabolites of nucleic acid metabolism, antibiotics,pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides,amines, amino acids, triazol nucleosides, or corticosteroids. Such otheragents may be present in the composition being administered or may beadministered separately. Also, the antibody is suitably administeredserially or in combination with radiological treatments, whetherinvolving irradiation or administration of radioactive substances.

The antibodies of the invention may be used as affinity purificationagents. In this process, the antibodies are immobilized on a solid phasesuch a Sephadex resin or filter paper, using methods well known in theart. The immobilized antibody is contacted with a sample containing theVEGF protein (or fragment thereof) to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the VEGF protein, which is boundto the immobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theVEGF protein from the antibody.

Anti-VEGF antibodies may also be useful in diagnostic assays for VEGFprotein, e.g., detecting its expression in specific cells, tissues, orserum. Such diagnostic methods may be useful in cancer diagnosis.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety. Numerous labels are available which can begenerally grouped into the following categories:

(a) Radioisotopes, such as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S.The antibody can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Volumes 1 and 2, Coligenet al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), forexample, and radioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,.beta.-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981). Examples ofenzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate aschromogenic substrate; and

(iii) .beta.-D-galactosidase (.beta.-D-Gal) with a chromogenic substrate(e.g., P-nitrophenyl-.beta.-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-.beta.-D-galactosidase.

In another embodiment of the invention, the anti-VEGF antibody need notbe labeled, and the presence thereof can be detected using a labeledantibody which binds to the VEGF antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of VEGF protein in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

For immunohistochemistry, the tumor sample may be fresh or frozen or maybe embedded in paraffin and fixed with a preservative such as formalin,for example.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radio nuclide (such as ¹¹¹In,⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

The antibody of the present invention can be provided in a kit, apackaged combination of reagents in predetermined amounts withinstructions for performing the diagnostic assay. Where the antibody islabeled with an enzyme, the kit will include substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides thedetectable chromophore or fluorophore). In addition, other additives maybe included such as stabilizers, buffers (e.g., a block buffer or lysisbuffer) and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will provide areagent solution having the appropriate concentration.

Pharmaceutical Preparations

In one aspect the invention provides pharmaceutical formulationscomprising anti-VEGF antibodies for the treatment of VEGF-mediateddiseases. The term “pharmaceutical formulation” refers to preparationswhich are in such form as to permit the biological actvity of theantibody or antibody derivative to be unequivocally effective, and whichcontain no additional components which are toxic to the subjects towhich the formulation would be administered. “Pharmaceuticallyacceptable” excipients (vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

A “stable” formulation is one in which the antibody or antibodyderivative therein essentially retains its physical stability and/orchemical stability and/or biological activity upon storage. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. Preferably, the formulation is stable at room temperature (about30° C.) or at 40° C. for at least 1 week and/or stable at about 2-8° C.for at least 3 months to 2 years. Furthermore, the formulation ispreferably stable following freezing (to, e.g., −70° C.) and thawing ofthe formulation.

An antibody or antibody derivative “retains its physical stability” in apharmaceutical formulation if it meets the defined releasespecifications for aggregation, degradation, precipitation and/ordenaturation upon visual examination of color and/or clarity, or asmeasured by UV light scattering or by size exclusion chromatography, orother suitable art recognized methods.

An antibody or antibody derivative “retains its chemical stability” in apharmaceutical formulation, if the chemical stability at a given time issuch that the protein is considered to still retain its biologicalactivity as defined below. Chemical stability can be assessed bydetecting and quantifying chemically altered forms of the protein.Chemical alteration may involve size modification (e.g. clipping) whichcan be evaluated using size exclusion chromatography, SDS-PAGE and/ormatrix-assisted laser desorption ionization/time-of-flight massspectrometry (MALDI/TOF MS), for example. Other types of chemicalalteration include charge alteration (e.g. occurring as a result ofdeamidation) which can be evaluated by ion-exchange chromatography, forexample.

An antibody or antibody derivative “retains its biological activity” ina pharmaceutical formulation, if the biological activity of the antibodyat a given time is within about 10% (within the errors of the assay) ofthe biological activity exhibited at the time the pharmaceuticalformulation was prepared as determined in an antigen binding assay, forexample. Other “biological activity” assays for antibodies areelaborated herein below.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and non-reducing sugars), sugar alcohols and sugaracids. Preferred polyols herein have a molecular weight which is lessthan about 600 kD (e.g. in the range from about 120 to about 400 kD). A“reducing sugar” is one which contains a hemiacetal group that canreduce metal ions or react covalently with lysine and other amino groupsin proteins and a “non-reducing sugar” is one which does not have theseproperties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Non-reducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof. Where it is desired that the formulation is freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes theantibody in the formulation. Non-reducing sugars such as sucrose andtrehalose are the preferred polyols herein, with trehalose beingpreferred over sucrose, because of the superior solution stability oftrehalose.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention has a pH in the range from about 4.5 to about8.0; preferably from about 5.5 to about 7. Examples of buffers that willcontrol the pH in this range include acetate (e.g. sodium acetate),succinate (such as sodium succinate), gluconate, histidine, citrate andother organic acid buffers. Where a freeze-thaw stable formulation isdesired, the buffer is preferably not phosphate.

In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” of an antibody or antibody derivativerefers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody or antibody derivativeis effective. A “disease/disorder” is any condition that would benefitfrom treatment with the antibody or antibody derivative. This includeschronic and acute disorders or diseases including those pathologicalconditions which predispose the mammal to the disorder in question.

A “preservative” is a compound which can be included in the formulationto essentially reduce bacterial action therein, thus facilitating theproduction of a multi-use formulation, for example. Examples ofpotential preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride (a mixture ofalkylbenzyldimethylammonium chlorides in which the alkyl groups arelong-chain compounds), and benzethonium chloride. Other types ofpreservatives include aromatic alcohols such as phenol, butyl and benzylalcohol, alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferredpreservative herein is benzyl alcohol.

The present invention also provides pharmaceutical compositionscomprising one or more antibodies or antibody derivative compounds,together with at least one physiologically acceptable carrier orexcipient. Pharmaceutical compositions may comprise, for example, one ormore of water, buffers (e.g., neutral buffered saline or phosphatebuffered saline), ethanol, mineral oil, vegetable oil,dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, adjuvants, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione and/or preservatives. As noted above, other activeingredients may (but need not) be included in the pharmaceuticalcompositions provided herein.

A carrier is a substance that may be associated with an antibody orantibody derivative prior to administration to a patient, often for thepurpose of controlling stability or bioavailability of the compound.Carriers for use within such formulations are generally biocompatible,and may also be biodegradable. Carriers include, for example, monovalentor multivalent molecules such as serum albumin (e.g., human or bovine),egg albumin, peptides, polylysine and polysaccharides such asaminodextran and polyamidoamines. Carriers also include solid supportmaterials such as beads and microparticles comprising, for example,polylactate polyglycolate, poly(lactide-co-glycolide), polyacrylate,latex, starch, cellulose or dextran. A carrier may bear the compounds ina variety of ways, including covalent bonding (either directly or via alinker group), noncovalent interaction or admixture.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical, intraocular, oral,nasal, rectal or parenteral administration. In certain embodiments,compositions in a form suitable for topical use, for example, as eyedrops, are preferred. Other forms include, for example, pills, tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups or elixirs. Withinyet other embodiments, compositions provided herein may be formulated asa lyophilizate. The term parenteral as used herein includessubcutaneous, intradermal, intravascular (e.g., intravenous),intramuscular, spinal, intracranial, intrathecal and intraperitonealinjection, as well as any similar injection or infusion technique.

The pharmaceutical composition may be prepared as a sterile injectibleaqueous or oleaginous suspension in which the modulator, depending onthe vehicle and concentration used, is either suspended or dissolved inthe vehicle. Such a composition may be formulated according to the knownart using suitable dispersing, wetting agents and/or suspending agentssuch as those mentioned above. Among the acceptable vehicles andsolvents that may be employed are water, 1,3-butanediol, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils may be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectible compositions, and adjuvants such aslocal anesthetics, preservatives and/or buffering agents can bedissolved in the vehicle.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of modulator following administration). Such formulations maygenerally be prepared using well known technology and administered by,for example, oral, rectal, or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofmodulator release. The amount of an antibody or antibody derivativecontained within a sustained release formulation depends upon, forexample, the site of implantation, the rate and expected duration ofrelease and the nature of the disease/disorder to be treated orprevented.

Antibody or antibody derivatives provided herein are generallyadministered in an amount that achieves a concentration in a body fluid(e.g., blood, plasma, serum, CSF, synovial fluid, lymph, cellularinterstitial fluid, tears or urine) that is sufficient to detectablybind to VEGF and prevent or inhibit VEGF-mediated diseases/disorders. Adose is considered to be effective if it results in a discerniblepatient benefit as described herein. Preferred systemic doses range fromabout 0.1 mg to about 140 mg per kilogram of body weight per day (about0.5 mg to about 7 g per patient per day), with oral doses generallybeing about 5-20 fold higher than intravenous doses. The amount ofantibody or antibody derivative that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Dosage unitforms will generally contain between from about 1 mg to about 500 mg ofan active ingredient.

Pharmaceutical compositions may be packaged for treating conditionsresponsive to an antibody or antibody derivative directed to VEGF.Packaged pharmaceutical compositions may include a container holding aeffective amount of at least one antibody or antibody derivative asdescribed herein and instructions (e.g., labeling) indicating that thecontained composition is to be used for treating a disease/disorderresponsive to one antibody or antibody derivative followingadministration in the patient.

The antibodies or antibody derivatives of the present invention can alsobe chemically modified. Preferred modifying groups are polymers, forexample an optionally substituted straight or branched chain polyalkene,polyalkenylene, or polyoxyalkylene polymer or a branched or unbranchedpolysaccharide. Such effector group may increase the half-live of theantibody in vivo. Particular examples of synthetic polymers includeoptionally substituted straight or branched chain poly(ethyleneglycol)(PEG), poly(propyleneglycol), poly(vinylalcohol) or derivatives thereof.Particular naturally occurring polymers include lactose, amylose,dextran, glycogen or derivatives thereof. The size of the polymer may bevaried as desired, but will generally be in an average molecular weightrange from 500 Da to 50000 Da. For local application where the antibodyis designed to penetrate tissue, a preferred molecular weight of thepolymer is around 5000 Da. The polymer molecule can be attached to theantibody, in particular to the C-terminal end of the Fab fragment heavychain via a covalently linked hinge peptide as described in WO0194585.Regarding the attachment of PEG moieties, reference is made to“Poly(ethyleneglycol) Chemistry, Biotechnological and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York and“Bioconjugation Protein Coupling Techniques for the BiomedicalSciences”, 1998, M. Aslam and A. Dent, Grove Publishers, New York.

After preparation of the antibody or antibody derivative of interest asdescribed above, the pharmaceutical formulation comprising it isprepared. The antibody to be formulated has not been subjected to priorlyophilization and the formulation of interest herein is an aqueousformulation. Preferably the antibody or antibody derivative in theformulation is an antibody fragment, such as an scFv. Thetherapeutically effective amount of antibody present in the formulationis determined by taking into account the desired dose volumes andmode(s) of administration, for example. From about 0.1 mg/ml to about 50mg/ml, preferably from about 0.5 mg/ml to about 40 mg/ml and mostpreferably from about 10 mg/ml to about 20 mg/ml is an exemplaryantibody concentration in the formulation.

An aqueous formulation is prepared comprising the antibody or antibodyderivative in a pH-buffered solution The buffer of this invention has apH in the range from about 4.5 to about 8.0, preferably from about 5.5to about 7. Examples of buffers that will control the pH within thisrange include acetate (e.g. sodium acetate), succinate (such as sodiumsuccinate), gluconate, histidine, citrate and other organic acidbuffers. The buffer concentration can be from about 1 mM to about 50 mM,preferably from about 5 mM to about 30 mM, depending, for example, onthe buffer and the desired isotonicity of the formulation.

A polyol, which acts as a tonicifier and may stabilize the antibody, isincluded in the formulation. In preferred embodiments, the formulationdoes not contain a tonicifying amount of a salt such as sodium chloride,as this may cause the antibody or antibody derivative to precipitateand/or may result in oxidation at low pH. In preferred embodiments, thepolyol is a non-reducing sugar, such as sucrose or trehalose. The polyolis added to the formulation in an amount which may vary with respect tothe desired isotonicity of the formulation. Preferably the aqueousformulation is isotonic, in which case suitable concentrations of thepolyol in the formulation are in the range from about 1% to about 15%w/v, preferably in the range from about 2% to about 10% whv, forexample. However, hypertonic or hypotonic formulations may also besuitable. The amount of polyol added may also alter with respect to themolecular weight of the polyol. For example, a lower amount of amonosaccharide (e.g. mannitol) may be added, compared to a disaccharide(such as trehalose).

A surfactant is also added to the antibody or antibody derivativeformulation. Exemplary surfactants include nonionic surfactants such aspolysorbates (e.g. polysorbates 20, 80 etc) or poloxamers (e.g.poloxamer 188). The amount of surfactant added is such that it reducesaggregation of the formulated antibody/antibody derivative and/orminimizes the formation of particulates in the formulation and/orreduces adsorption. For example, the surfactant may be present in theformulation in an amount from about 0.001% to about 0.5%, preferablyfrom about 0.005% to about 0.2% and most preferably from about 0.01% toabout 0.1%.

In one embodiment, the formulation contains the above-identified agents(i.e. antibody or antibody derivative, buffer, polyol and surfactant)and is essentially free of one or more preservatives, such as benzylalcohol, phenol, m-cresol, chlorobutanol and benzethonium Cl. In anotherembodiment, a preservative may be included in the formulation,particularly where the formulation is a multidose formulation. Theconcentration of preservative may be in the range from about 0.1% toabout 2%, most preferably from about 0.5% to about 1%. One or more otherpharmaceutically acceptable carriers, excipients or stabilizers such asthose described in Remington's Pharmaceutical Sciences 21st edition,Osol, A. Ed. (2006) may be included in the formulation provided thatthey do not adversely affect the desired characteristics of theformulation. Acceptable carriers, excipients or stabilizers arenon-toxic to recipients at the dosages and concentrations employed andinclude: additional buffering agents, co-solvents, antioxidantsincluding ascorbic acid and methionine, chelating agents such as EDTA,metal complexes (e.g. Zn-protein complexes), biodegradable polymers suchas polyesters, and/or salt-forming counterions such as sodium.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, preparation of the formulation.

The formulation is administered to a mammal in need of treatment withthe antibody, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. In preferred embodiments, the formulationis administered to the mammal by topical application of eye drops to theocular surface. For such purposes, the formulation may applied using aneye drop applicator, for example.

The appropriate dosage (“therapeutically effective amount”) of theantibody will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, the type ofantibody used, and the discretion of the attending physician. Theantibody or antibody derivative is suitably administered to the patentat one time or over a series of treatments and may be administered tothe patent at any time from diagnosis onwards. The antibody or antibodyderivative may be administered as the sole treatment or in conjunctionwith other drugs or therapies useful in treating the condition inquestion.

As a general proposition, the therapeutically effective amount of theantibody or antibody derivative administered will be in the range ofabout 0.1 to about 50 mg/kg of patent body weight whether by one or moreadministrations, with the typical range of antibody used being about 0.3to about 20 mg/kg, more preferably about 0.3 to about 15 mg/kg,administered daily, for example. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques.

FDA approved doeses and regimes suitable for use with Lucentis areconsidered.

Other doses and regimes are described in U.S. Provisional ApplicationSer. No. 61/075,641, entitled “Improved Immunobinder Formulations AndMethods For Administration”, filed Jun. 25, 2008, which is expresslyincorporated herein.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the aqueous pharmaceuticalformulation of the present invention and optionally providesinstructions for its use. Suitable containers include, for example,bottles, vials, eye drop applicators and syringes. The container may beformed from a variety of materials such as glass or plastic. Anexemplary container is a 3-20 cc single use glass or plastic vial.Alternatively, for a multidose formulation, the container may be 3-100cc glass vial. The container holds the formulation and the label on, orassociated with, the container may indicate directions for use. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

EXEMPLIFICATION

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference in their entireties.

Throughout the examples, the following materials and methods were usedunless otherwise stated.

General Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques of polypeptidepreparation. See, e.g., Sambrook, Fritsch and Maniatis, MolecularCloning: Cold Spring Harbor Laboratory Press (1989); AntibodyEngineering Protocols (Methods in Molecular Biology), 510, Paul, S.,Humana Pr (1996); Antibody Engineering: A Practical Approach (PracticalApproach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: ALaboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); andCurrent Protocols in Molecular Biology, eds. Ausubel et al., John Wiley& Sons (1992).

Thermostability Measurements

Attenuated total reflectance Fourier transform IR (FTIR-ATR) spectrawere obtained for various single chains and derivative molecules usingthe FT-IR Bio-ATR cell in a Tensor Bruker. The molecules wereconcentrated up to 3 mg/ml and dialyzed overnight at 4° C. against PBS,pH 6.5 and the buffer flow through was collected as blank. Thedenaturation profiles were obtained by thermo challenging the moleculeswith a broad range of temperatures in 5° C. steps (25 to 95° C.). Allspectra manipulations were performed using OPUS software. The mainbuffer and transient atmospheric (CO₂ and H₂O) background weresubstracted from the protein spectrum. The resulting protein spectrumwas then baseline corrected and the protein amide I spectra wasdetermined from the width of the widest resolvable peak in the expectedregion. Second derivative spectra were obtained for the amide I bandspectra using a third degree polynomial function with a smoothingfunction. Changes in protein structure were estimated by amide I secondderivative analysis using a linear calibration curve for the initialcurve-fit calculations assuming 0% denaturation for the 3 lowermeasurements and 100% denaturation for the 3 higher measurements. Thedenaturation profiles were used to approximate midpoints of the thermalunfolding transitions (TM) for every variant applying the Boltzmannsigmoidal model.

Solubility Measurements

Relative solubility of various scFv molecules was measured afterenhancing protein aggregation and precipitation in presence of ammoniumsulfate. Ammonium sulfate was added to the protein in aqueous solutionsto yield increments of 5% of saturation in the final mixturesalt-protein. The precipitation in the dynamic range was determinedempirically and the saturation intervals reduced in this range to 2.5%intervals saturation in the final mixture. After ammonium sulfateaddition, samples were gently mixed and centrifuged 30 minutes at 6000rpm. The remaining protein in supernatants was recovered for eachammonium sulfate percentage of saturation. Solubility curves weredetermined by measuring the protein concentration in the supernatant byUV-VIS measurements using NanoDrop™ 1000 Spectrophotometer. Measurementsof remaining soluble protein in supernatants were normalized and used toestimate midpoints of relative solubility for every variant applying theBoltzmann sigmoidal model.

Short Term Stability Test

The scFv molecules were examined after two weeks incubation at 40° C.for the presence of soluble aggregates and degradation products.Proteins with a concentration of 10 mg/ml were dialyzed overnight at 4°C. against PBS with a broad range of pHs (3.5, 4.5, 5.5, 6.5, 7.0, 7.5and 8.5). Control molecules with the same concentration in standardbuffer PBS (pH 6.5) were stored at −80° C. during the 2 weeks period.Determination of degradation bands by SDS-PAGE was done at t=0 and t=14dtime points and soluble aggregates were assessed in the SEC-HPLC.Determination of remaining activity after 2 weeks at 40° C. was doneusing Biacore.

Example 1 Immunization Strategy for Generating Anti-VEGF Antibodies

In this example, an immunization strategy is described which used anovel antigenic VEGF-derived peptide, to generate antibodies capable ofrecognizing human, mouse and rabbit VEGFA.

From alanine-scanning mutagenesis studies performed at Genentech theresidues of VEGFA that are crucial for high affinity interaction withVEGFr are known (Fuh, G. et al, (2006) J. Biol. Chem. 281, 6625-6631).Although the receptor-binding site probably represents a conformationalepitope, most of the crucial residues lie on an alpha helix, on thefirst 10 amino acids of mature VEGFA.

Rabbit VEGFA contains three amino acids changes in this alpha helix,when compared to the human sequence; in contrast, mouse VEGFA isidentical to human in this region. Thus, for the generation ofmouse-human cross-reactive antibodies, rabbit presents a suitablespecies for immunization. In addition, rabbit immunization can lead toAbs with higher affinity than mouse immunization.

As outlined above, interaction with residues on the N-terminal alphahelix of VEGFA seems to be most crucial for binding to VEGFR1.Therefore, this 10 amino acid long stretch can be used as an epitope forimmunization. Alternatively, full length VEGFA can be injected, however,other peptide stretches on VEGFA are more immunogenic, thus lowering thechance to raise neutralizing antibodies. This hypothesis is supported bythe fact that two different peptides, both lying close to the C-terminusof VEGFA are potentially immunogenic as predicted by the method ofJohnson and Wolf. This method predicts only minor immunogenic potentialfor the N-terminal alpha helix. Therefore, immunization with the peptideconstituting the alpha helix only, can be more straightforward thanimmunization with full-length VEGFA. The probability to elicit a strongimmune response can be further increased by fusion or chemical couplingof the peptide to Keyhole Limpet Hemocyanin (KLH).

Four immunization strategies were performed as follows

A. Pre-Immunization of rabbits with full-length human VEGFA₁₆₅ toenhance the probability to obtain conformational binders. Second boostwith peptide from aa stretch 16-K FMD V YQ RS Y CHP-28 (underline:receptor interaction; double underline, divergent in rabbit, Cys isinvolved in disulfide bond according to crystal structure). The Cyscontained in the peptided sequence could be used for coupling to KLH andwould therefore not be exposed as free Cys. The final peptide would lookas follows: KFMDVYQRSY-Cys-KLH.B. Pre-Immunization of mice with full-length VEGFA₁₆₅ to enhance theprobability to obtain conformational binders. Second boost with peptidefrom aa stretch 16-K FMD V YQ RS Y CHP-28 (Cys is involved in disulfidebond according to crystal structure). The Cys contained in the peptidedsequence can be used for coupling to KLH and would therefore not beexposed as free Cys. The final peptide would look as follows:KFMDVYQRSY-Cys-KLH.C. Pre-immunization of rabbits/mice with peptide from aa stretch 16-KFMD V YQ RS Y CHP-28 (final peptide: KFMDVYQRSY-Cys-KLH). Second boostwith full-length VEGFA₁₆₅ to enhance the probability to obtainconformational binders.D. Immunization with full length VEGFA₁₆₅ in rabbits.

Example 2 CDR Grafting and Functional Humanization of Monoclonal RabbitAnti-VEGF Antibodies

Grafting of Rabbit CDRs

Unlike traditional humanization methods which employ the human antibodyacceptor framework that shares the greatest sequence homology with thenon-human donor antibody, the rabbit CDRs were grafted into eitherframework FW1.4 (SEQ ID No. 172) to generate a Min-graft or into the“rabbitized” framework rFW1.4 (SEQ ID No. 173) or its variant rFW1.4(v2)(SEQ ID No. 174) to generate a Max-graft. Both frameworks were selectedprimarily for desirable functional properties (solubility andstability), structural suitability to accommodate a large variety ofrabbit CDRs and reasonable homology to the rabbit variable domainconsensus sequence. Framework rFW1.4 is a derivative of FW1.4 that wasfurther engineered with the aim to serve as universal acceptor frameworkfor virtually any set of rabbit CDRs. Although the stable and solubleframework sequence FW1.4 exhibits high homology to rabbit antibodies, itis not the most homologous sequence available.

Identification of Residues Potentially Involved in Binding

For each rabbit variable domain sequence, the nearest rabbit germlinecounterpart was identified. If the closest germline could not beestablished, the sequence was compared against the subgroup consensus orthe consensus of rabbit sequences with a high percentage of similarity.Rare framework residues were considered as possible result of somatichypermutation and therefore playing a role in antigen binding.Consequently, such residues were considered for grafting onto theacceptor framework rFW1.4 or rFW1.4(v2) to generate Max-grafts.Particularly, residues potentially implicated in direct antigen contactor influencing disposition of VL and VH were grafted. Further residuesdescribed to influence CDR structure were substituted if required. Noframework substitutions were made when CDRs were grafted onto FW1.4(Min-grafts). For example to generate 578minmax residue VH 94 (H94) ofrFW1.4 was mutated to corresponding residue in the donor sequence. Therabbit antibody 578 contains Gly at H94 whereas both, the mosthomologous germline and the rabbit consensus contain Arg at positionH94. Gly has an exceptional flexibility (positive phi angles) that isnot found for other amino acids. This suggests a role in mainchaintorsion angle and a possible strong influence of the loop conformationwith implications on activity. Further examples of framework positionsthat were grafted to obtain the Max-grafts as disclosed herein can beidentified by making a sequence alignment of the framework regions ofrFW1.4, rFW1.4(v2) and the scFv sequences of interest provided herein.Webtools as known in the art may for example be used for said purpose(e.g. ClustalW as available on Jun. 23, 2009 athttp://www.ebi.ac.uk/Tools/clustalw2/index.html or MultiAlin asavialable on Jun. 23, 2009 at http://bioinfo.genotoul.fr/multalin). Allframework positions at which rFW1.4 and rFW1.4(v2) contain the sameresidue and at which the scFv of interest reveals a different residue,are framework positions that were grafted to obtain the Max-grafts.

Domain Shuffling

Variable light chains of Min-grafts were combined with variable heavychain Max-grafts to identify optimal combinations in terms ofbiophysical properties (solubility and stability) and activity.

Cloning and Expression of scFvs

The scFvs described and characterized herein were produced as follows.The humanized VL sequences (SEQ ID NOs:82-106) were connected tohumanized VH sequences (SEQ ID NOs:118-166) via the linker of SEQ IDNO:181 to yield an scFv of the following orientation:NH₂-VL-linker-VH-COOH. In many cases DNA sequences encoding for thevarious scFvs were de novo synthesized at the service providerEntelechon GmbH (www.entelechon.com). The resulting DNA inserts werecloned into the bacterial expression vector pGMP002 via NcoI and HindIIIrestriction sites introduced at the 5′ and 3′ end of the scFv DNAsequence, respectively. Between the DNA sequence of the VL domain andthe VH domain, a BamHI restriction site is located. In some cases thescFv encoding DNA was not de novo synthesized, but the scFv expressingconstructs were cloned by domain shuffling. Accordingly, the VL domainswere excised and introduced into the new constructs via NcoI and BamHIrestriction sites, the VH domains via BamHI and HindIII restrictionsites. In other cases, point mutations were introduced into the VHand/or VL domain using state of the art assembling PCR methods. Thecloning of GMP002 is described in Example 1 of WO2008006235. Theproduction of the scFvs was done analogue as for ESBA105 as described inExample 1 of WO2008006235.

Example 3 Biacore Binding Analysis of Anti-VEGF scFvs

In this example, the Biacore-binding ability of scFvs was tested and thebinding affinity was measured using the exemplary surface plasmonresonance method with BIAcore™-T100. The VEGF proteins, tested forbinding by these scFv candidates, in this example and later examplesinclude purified Escherichia coli-expressed recombinant human VEGF₁₆₅(PeproTech EC Ltd.), recombinant human VEGF₁₂₁ (PeproTech EC Ltd.),recombinant human VEGF₁₁₀ (ESBATech AG), recombinant murine VEGF₁₆₄(PeproTech EC Ltd.), recombinant rat VEGF₁₆₄ (Biovision), recombinantrabbit VEGF₁₁₀ (ESBATech AG), and recombinant human PLGF (PeproTech ECLtd.). For the surface plasmon resonance experiment, carboxymethylateddextran biosensor chips (CM4, GE Healthcare) were activated withN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide according to the supplier's instructions. Each ofthe 6 different VEGF forms, as exemplified above, was coupled to 1 ofthe 4 different flow cells on a CM4 sensor chip using a standardamine-coupling procedure. The range of responses obtained with theseimmobilized VEGF molecules after coupling and blocking were ˜250-500response units (RU) for hVEGF₁₆₅, ˜200 RU for hVEGF₁₁₀, hVEGF₁₂₁, murineVEGF₁₆₄, rat VEGF₁₆₄ and rabbit VEGF₁₁₀ and ˜400 RU for PLGF. The 4thflow cell of each chip was treated similarly except no proteins wereimmobilized prior to blocking, and the flow cell was used as in-linereference. Various concentrations of anti-VEGF scFvs (e.g., 90 nM, 30nM, 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM and 0.04 nM) in HBS-EPbuffer (0.01 M HEPES, pH 7.4 or 5, 0.15 M NaCl, 3 mM EDTA, 0.005%surfactant P20) were injected into the flow cells at a flow rate of 30μl/min for 5 min. Dissociation of the anti-VEGF scFv from the VEGF onthe CM4 chip was allowed to proceed for 10 min at 25° C. Sensorgramswere generated for each anti-VEGF scFv sample after in-line referencecell correction followed by buffer sample subtraction. The apparentdissociation rate constant (k_(d)), the apparent association rateconstant (k_(a)) and the apparent dissociation equilibrium constant(K_(D)) were calculated using one-to-one Langmuir binding model withBIAcore T100 evaluation Software version 1.1.

As one exemplary result, some lead anti-VEGF scFv candidates are listedin Table 7 showing their binding affinity to hVEGF₁₆₅. Their potency asVEGF inhibitors, which is measured using VEGFR competition ELISA and/orHUVEC assay and described in latter examples, is also shown in Table 7.The kinetics curves of some exemplary lead candidates, e.g., 511max and578max, for their binding to hVEGF₁₆₅ are illustrated in FIG. 1. Theiraffinity constants (k_(d), k_(a) and K_(D)) were also determined. Somelead candidates also display species specificity in their binding tovarious VEGF proteins of different sources. For example, some affinitydata measured at pH5 using mouse and rat VEGF₁₆₄ as binding partner areshown in Tables 8 a and b. An exemplary lead scFv candidate, 578minmax,has a K_(D) of 5.76E-10 M and 7.48E-10 M in its binding to mouse and ratVEGF₁₆₄, respectively at a pH of 5 (Tables 8 a and b) and 2.73E-11 and2.19E-11 at a pH of 7.4 (data not shown). This species specificity isfurther illustrated in FIG. 4 in the kinetics curves and affinity datafor the binding between 578minmax and human, mouse or rat VEGF proteins.

Besides the species specificity in their binding to VEGFs from differentorganisms, many lead scFv candidates also display differentiated bindingaffinities towards various VEGF isoforms. For example, the affinity datameasured at pH 5.0 for some scFv candidates binding to human VEGF₁₆₅,VEGF₁₂₁ and VEGF₁₁₀ are compared in Table 9. In the same experiments,PIGF protein was also used as a negative control without bindingcapacity to those scFv candidates. Also, the differentiated kineticscurves and affinity data for the binding between 578Max and VEGFisoforms, as an example, are illustrated in FIG. 3.

The present invention also discloses derivatives originating from thelead anti-VEGF scFv candidates, which are mentioned above. Some leadderivatives of candidate 578 and 511, as listed in Table 10, areexemplified for their affinity and potency (measured at pH 5.0). In thisexperiment, Biacore measurement was used for the affinity of thesederivatives towards hVEGF₁₆₅, while hVEGFR2 competition ELISA and/orHUVEC assay were used to define their potency to inhibit VEGFs (Table10). Three derivatives, 578max, 578minmax and 578 wt-His, are furtherexemplified in their kinetics curves and affinity data for binding tohVEGF₁₆₅ in FIG. 4.

For derivatives of lead candidates, their biophysical characterizationswere determined and exemplified in FIGS. 5-7 and table 11. Thesecharacteristics include, as exemplified in table 11, T_(m) determined byFTIR, the percentage of β-sheet or protein loss after incubation at 60°C. for 30 min, solubility determined by ammonium sulfate precipitation,refolding yield during the production process and expression levels inE. coli. Three derivatives, 578max, 578minmax and 578minmax_DHP, werecharacterized for their thermal stability in their unfolding curvesagainst different temperatures measured by FT-IR (FIG. 5).

TABLE 7 overview of affinity and potency of lead candidates Rel.activity hVEGR2 Rel. activity hVEGR1 Rel. activity in HUVEC comp. ELISAcomp. ELISA assay Protein (EC50_(Luc)[nM]/ (EC50_(Luc)[nM]/(EC50_(Luc)[nM]/ ID Nr. EC50_(test)[nM]) EC50_(test)[nM])EC50_(test)[nM]) 375-min 857 0.3 ND ND 375-max 873 0.6 ND ND 509-min 8541.0 2.9 ND 509-max 855 4.1 13 0.003 509-maxII 856 0.6 0.09 0.0009511-min 801 4.9 0.7 0.0011 511-max 802 8.7 8 0.0179 534-min C-His 8070.1 ND ND 534-max 793 1.1 ND 0.0014 567-min 884 9.7 14.9/57   ND 567-max874 4.1 15.7/54.5 0.0086 578-min 820 4.1 4.8 0.1001 578-max 821 9.635.5/51.6 1.483 610-min 882 0.1 ND ND 610-max 883 0.4 ND ND 435-min 9440.03 ND ND 435-max 945 7.6 0.00039 ND Biacore Measurements (pH 5)Biacore Measurements (pH 7.4) hVEGF₁₆₅ hVEGF₁₆₅ ID ka (1/Ms) kd (1/s) KD(M) ka (1/Ms) kd (1/s) KD (M) 375-min 9.27E+05 5.01E−03 5.41E−09   >E+083.86E+00 NA 375-max 2.44E+06 6.55E−03 2.68E−09 5.09E+07 2.42E−014.74E−09 509-min 6.23E+05 1.14E−03 1.82E−09 3.52E+06 1.08E−02 3.06E−09509-max 2.26E+06 2.72E−03 1.21E−09 1.42E+06 5.37E−04 3.78E−10 509-maxII8.38E+05 2.82E−03 3.37E−09 7.59E+06 1.98E−02 2.61E−09 511-min 5.05E+051.28E−03 2.53E−09 6.75E+05 8.85E−04 1.31E−09 511-max 6.59E+05 4.40E−056.67E−11 8.00E+05 6.85E−05 8.56E−11 534-min C-His 2.71E+05 9.21E−033.41E−08 ND ND ND 534-max 1.88E+06 1.73E−02 9.21E−09 1.06E+06 2.62E−032.47E−09 567-min 2.01E+06 4.61E−04 2.30E−10 1.11E+06 7.00E−04 6.31E−10567-max 1.20E+06 2.26E−04 1.88E−10 1.17E+06 1.67E−04 1.43E−10 578-min1.14E+06 1.03E−02 9.01E−09 1.11E+06 2.02E−04 1.81E−10 578-max 7.00E+053.07E−04 4.39E−10 1.58E+06 3.76E−05 2.37E−11 610-min 2.51E+05 2.65E−031.06E−08 No No No binding binding binding 610-max 5.09E+05 6.01E−041.18E−09   >E+08 3.57E+01 NA 435-min No No No 4.95E+05 1.43E−02 2.89E−08binding binding binding 435-max 1.67E+05 7.55E−04 4.53E−09 1.13E+061.04E−04 9.22E−11

TABLE 8a species specificity of selected lead candidates (mouse and ratVEGF 164) Protein mouse VEGF₁₆₄ rat VEGF ID No. ka (1/Ms) kd (1/s) KD(M) ka (1/Ms) kd (1/s) KD (M) 509-min 854 6.14E+05 1.00E−03 1.63E−093.51E+05 8.44E−04 2.41E−09 509-max 855 4.09E+06 5.90E−03 1.45E−093.90E+06 6.45E−03 1.65E−09 509-maxII 856 3.47E+07 6.01E−02 1.73E−091.47E+07 2.66E−02 1.81E−09 511-min 801 6.25E+05 1.03E−03 1.64E−095.50E+05 1.12E−03 2.04E−09 511-max 802 7.53E+05 4.61E−05 6.13E−116.26E+05 6.63E−05 1.06E−10 567-min 884 2.06E+06 3.50E−04 1.70E−101.72E+06 4.80E−04 2.79E−10 567-max 874 1.64E+06 1.52E−04 9.29E−111.36E+06 2.03E−04 1.49E−10 578-min 820 1.40E+06 1.51E−02 1.07E−081.70E+06 1.82E−02 1.07E−08 578-max 821 1.03E+06 4.40E−04 4.29E−108.83E+05 5.28E−04 5.98E−10

TABLE 8b species specificity of selected development candidates Relativevalues Biacore measurements Mouse VEGF₁₆₄ Protein mouse VEGF₁₆₄ (kdh_(VEGF165)/ (Kd_(hVEGF165)/ ID No. ka (1/Ms) kd (1/s) KD (M)kd_(mVEGF164)) Kd_(mVEGF164)) 578minmax 903 1.14E+06 6.57E−04 5.67E−100.8 1.1 578 minmax_FW1.4: DHP 961 1.10E+06 6.69E−04 6.08E−10 0.6 0.9578minmaxT84N_V89L 1008 1.23E+06 1.88E−03 1.53E−09 1.0 1.0 578min_max1017 1.47E+06 2.16E−03 1.46E−09 1.4 1.8 T84N_V89L_DHP Relative valuesBiacore measurements Mouse VEGF₁₆₄ Protein rat VEGF₁₆₄ (kd h_(VEGF165)/(Kd_(hVEGF165)/ ID No. ka (1/Ms) kd (1/s) KD (M) kd_(mVEGF164))Kd_(mVEGF164)) 578minmax 903 8.58E+05 6.41E−04 7.48E−10 0.8 0.8 578minmax_FW1.4: DHP 961 8.00E+05 6.76E−04 8.45E−10 0.6 0.7578minmaxT84N_V89L 1008 8.02E+05 1.52E−03 1.89E−09 1.2 0.8 578min_max1017 1.04E+05 1.90E−03 1.82E−09 1.6 1.5 T84N_V89L_DHP

TABLE 9 Binding of selected lead candidates to VEGF isoforms (humanVEGF121 and hVEGF110) Protein hVEGF₁₆₅ hVEGF₁₁₀ ID Nr. ka (1/Ms) kd(1/s) KD (M) ka (1/Ms) kd (1/s) KD (M) 509-min 854 6.23E+05 1.14E−031.82E−09 2.87E+05 4.74E−04 1.65E−09 509-max 855 2.26E+06 2.72E−031.21E−09 6.48E+05 2.35E−04 3.63E−10 509-maxII 856 8.38E+05 2.82E−033.37E−09 9.01E+05 1.33E−03 1.48E−09 511-min 801 5.05E+05 1.28E−032.53E−09 6.19E+05 8.98E−04 1.45E−09 511-max 802 6.59E+05 4.40E−056.67E−11 4.05E+05 7.96E−05 1.97E−10 567-min 884 2.01E+06 4.61E−042.30E−10 1.52E+06 3.82E−05 2.51E−11 567-max 874 1.20E+06 2.26E−041.88E−10 1.00E+06 3.27E−05 3.27E−11 578-min 820 1.14E+06 1.03E−029.01E−09 9.15E+05 1.04E−02 1.14E−08 578-max 821 7.00E+05 3.07E−044.39E−10 5.23E+05 7.22E−04 1.38E−09 hVEGF₁₂₁ ID ka (1/Ms) kd (1/s) KD(M) PlGF 509-min 3.54E+05 4.53E−04 1.28E−09 no binding 509-max 7.42E+052.49E−04 3.35E−10 no binding 509-maxII 8.97E+05 1.23E−03 1.37E−09 nobinding 511-min 7.78E+05 9.63E−04 1.24E−09 no binding 511-max 4.67E+059.97E−05 2.14E−10 no binding 567-min 1.89E+06 4.54E−05 2.41E−11 nobinding 567-max 1.13E+06 5.76E−05 5.11E−11 no binding 578-min 9.61E+058.80E−03 9.16E−09 no binding 578-max 5.87E+05 5.58E−04 9.50E−10 nobinding

TABLE 10 Overview on affinity and potency of lead derivatives (578 and511) Rel. activity Rel. activity in hVEGR2 HUVEC assay comp. ELISA(EC50_(Luc)[nM]/ Biacore Measurements Protein (EC50_(Luc)[nM]/EC50_(test)[nM]) hVEGF₁₆₅ ID Nr. EC50_(test)[nM]) hVEGF ka (1/Ms) kd(1/s) KD (M) 578 wildtype C-His 798 ND ND 8.34E+05 1.69E−04 2.00E−10578-min 820 4.1 0.1001 1.14E+06 1.03E−02 9.01E−09 578-max 821 9.60.94/1.0/1.2/1.2 7.00E+05 3.07E−04 4.39E−10 (new setup) 578-maxFW1.4_DHP 960 ND ND 9.30E+05 2.48E−04 2.66E−10 578-minmax 903 8.4 1.6/1.4 8.06E+05 5.04E−04 6.25E−10 (new setup) 578minmax 961 16.5 0.78/1.9 7.11E+05 4.09E−04 5.76E−10 FW1.4_DHP 578-max-min 902 6.5 ND1.35E+06 8.83E−03 6.55E−09 578min_max T84N 991 ND ND 7.21E+05 7.00E−049.71E−10 578min_max V89A 978 ND ND 5.09E+05 6.12E−04 1.20E−09 578min_maxV89L 980 ND ND 8.75E+05 1.87E−03 2.13E−09 578min_max 1008 8.4 ND1.13E+06 1.80E−03 1.59E−09 T84N_V89L 578min max 1009 7.5 ND 8.01E+054.93E−04 6.15E−10 T84N_V89A 578min max 1017 ND ND ND ND ND T84N_V89L_DHP578min_max ND ND ND ND ND T84N_V89A_DHP 578max synth FW opt 950 ND ND1.35E+06 5.86E−04 4.33E−10 578min_max_synthFW 997 7.2 ND 1.23E+069.89E−04 8.03E−10 578max_min_synthFW 990 ND ND 1.55E+06 5.31E−033.42E−09 578min_max_FW1. 1016 ND ND 7.08E+05 7.02E−04 9.91E−10 synth511-min 801 4.9 0.0011 5.05E+05 1.28E−03 2.53E−09 511-max 802 8.7 0.01796.59E+05 4.40E−05 6.67E−11 511min_max 904 5.4 ND 3.66E+05 1.02E−042.78E−10 511max_min 905 ND ND 5.11E+05 7.54E−04 1.48E−09

TABLE 11 Overview of biophysical characterization of lead derivatives(578 and 511) TM in %-sheet % Protein Bio- loss loss Protein ATR(Aquaspec (precipitation ID Nr. [° C.] 60° C.) at 60° C.) 578-min 82066.85 ND ND 578-max 821 70.36 −1.93% 16.20% 578-max FW1.4_DHP 960 ND NDND 578-minmax 903 71.12 −0.52% 10.99% 578minmax FW1.4_DHP 961 70.18−0.15% 14.82% 578-max-min 902 ND ND ND 578min_max T84N 991 70.78 0.11%20.30% 578min_max V89A 978 63.23 −2.28% 48.22% 578min_max V89L 980 68.15−0.79% 38.99% 578min_max 1008 69 −0.80% 28.30% T84N_V89L 578min_max 1009ND ND ND T84N_V89A 578min_max 1017 67.8 ND ND T84N_V89L_DHP 578min_max1080 66.3 ND ND T84N_V89A_DHP 578max synth FW opt 950 63.62 54.06%97.85% 578min_max_synthFW 997 63.25 50.89% 98.02% 578max_min_synthFW 990ND ND ND 578min_max_FW1. synth 1016 65.7 −0.20% 21.30% 511-min 801 ND NDND 511-max 802 70.5 −1.53% 4.50% 511min_max 904 ND ND ND 511max_min 905ND ND ND 567min 884 54 100.00% 100.00% Solubility by ammonium sulfateExpression precipitation Production: level in [EC₅₀ in % of Refolding E.coli NH₄(SO₄)₂ yield [arbitrary ID saturation] [mg/L] units] 578-min ND1.5 ++ 578-max 27.24 12.5 + 578-max FW1.4_DHP ND 11.6 + 578-minmax 28.1323.93 +++ 578minmax 32.36 50.5 +++ FW1.4_DHP 578-max-min ND 4.5 +578min_max T84N ND 7.5 +++ 578min_max V89A ND 16 +++ 578min_max V89L ND30 +++ 578min_max 27.88 24 +++ T84N_V89L 578min_max ND 22 +++ T84N_V89A578min_max 30.80 36 +++ T84N_V89L_DHP 578min_max 30.70 30 +++T84N_V89A_DHP 578max synth FW opt 28.30 19.4 ++ 578min_max_synthFW 30.0524 +++ 578max_min_synthFW ND 0.5 ++ 578min_max_FW1. 25.10 28 +++ synth511-min ND 13.5 +++ 511-max  8.62 6.47 +++ 511min_max ND 3.75 +++511max_min ND 7 +++ 567min 20.70 16.5 +++

Some derivatives, as listed in FIG. 6, were compared for theirdenaturation and precipitation after thermal stress (e.g., under 50° C.,60° C., or 70° C.) for 30 minutes. 578max, 578minmax and 578minmax_DHPwere further exemplified for their solubility, which was determined byammonium sulfate precipitation. As in FIG. 7, the percentage of solubleproteins of these derivatives under various concentrations of ammoniumsulfate were compared.

TABLE 12a Sample name Beta sheet % Nanodrop (mg/ml) anti-VEGF bindersafter incubation for 30 min at 50° C. 950 100.8 81.2 978 100.9 85.1 98099.9 100.3 991 99.4 99.2 802 100.4 96.7 821 100.6 93.5 903 99.5 99.4 96198.7 101.7 997 99.9 76.39 anti-VEGF binders after incubation for 30 minat 60° C. 950 45.9 2 978 102.3 52 980 100.8 61 991 99.9 80 802 101.5 96821 101.9 84 903 100.5 89 961 100.1 85 997 49.1 2 anti-VEGF bindersafter incubation for 30 min at 70° C. 950 43.1 1.0 978 13.4 2.7 980 4.50.2 991 21.5 1.4 802 100.4 80.8 821 58.4 3.3 903 81.9 0.7 961 46.3 1.1997 0.0 0.3

Example 4 VEGF Receptor Blocking Assays

For anti-VEGF scFv candidates or their derivatives disclosed in thepresent invention, their potency as VEGF inhibitors was also measuredbesides their binding affinity to VEGFs in Example 3. The methods tomeasure their potency include, for example, the VEGFR competition ELISA,as exemplified in this example, and HUVEC assays (FIG. 8).

The VEGFR competition ELISA assays include, for example, VEGFR2 Receptorblocking assays and VEGFR1 Receptor blocking assays. For VEGFR2 Receptorblocking assay, human VEGF₁₆₅ was coated on a 96-well Maxisorp ELISAplate (Nunc) at 0.05 μg/ml in PBS and blocked using PBS with 0.1% BSAand 0.2% Tween 20 (PBST). 500 ng/ml recombinant human VEGFR2/Fc chimera(R&D Systems Inc.), consisting of amino acid residues 1-764 of theextracellular domain of human VEGFR2 fused to a 6× histidine tagged Fcof human IgG₁, was first incubated with 3-fold serially dilutedanti-VEGF scFvs in PBST. After 30-60 min of incubation at roomtemperature, the mixtures were transferred to the human VEGF₁₆₅immobilized plate and incubated for 90 min. Binding of the VEGFR2/Fcchimera to the immobilized VEGF₁₆₅ was detected with goat (Fab₂)anti-human IgG Fcγ coupled to horseradish peroxidase (JacksonImmunoResearch) followed by substrate (BM Blue POD substrate, RocheDiagnostics). Optical density at 450 nm (OD 450 nm) was measured using aSunrise microplate reader (Tecan). Data were analyzed using a4-parameter logistic curve fit, and EC₅₀ values were calculated from thedose-response curves of the scFvs. The exemplary potency of leadcandidates or their derivatives, measured by VEGFR2 Receptor blockingassay, is listed in Table 7 and 9.

For VEGFR1 Receptor blocking assay, human VEGF₁₆₅ was coated on a96-well Maxisorp ELISA plate (Nunc) at 0.0125 μg/ml in PBS and blockedusing PBS with 0.4% BSA and 0.1% Tween 20. 100 ng/ml of recombinanthuman VEGFR1/Fc chimera (R&D Systems Inc.), consisting of amino acidresidues 1-687 of the extracellular domain of human VEGFR1 fused to a 6×histidine tagged Fc of human IgG₁, was first incubated with 3-foldserially diluted anti-VEGF scFvs in PBST. After 30-60 min of incubationat room temperature, the mixtures were transferred to the human VEGF₁₆₅immobilized plate and incubated for 90 min. Binding of the VEGFR1/Fcchimera to the immobilized VEGF₁₆₅ was detected with goat (Fab₂)anti-human IgG Fcγ coupled to horseradish peroxidase (JacksonImmunoResearch) followed by substrate (BM Blue POD substrate, RocheDiagnostics). Optical density at 450 nm (OD 450 nm) was measured using aSunrise microplate reader (Tecan). Data were analyzed as above, and EC₅₀values were calculated from the dose-response curves of the scFvs. Theexemplary potency of lead candidates, measured by VEGFR1 Receptorblocking assay, is listed in Table 7.

Example 5 HUVEC Assay of VEGF Inhibition

This example exemplifies HUVEC assays as another method to measure thepotency of the disclosed anti-VEGF scFv candidates, or theirderivatives, as VEGF inhibitors.

Human umbilical vein endothelial cells (HUVECs) (Promocell), pooled fromseveral donors, were used at passage 2 to passage 14. Cells were seededat 1000 cells/well in 50 μl complete endothelial cell growth medium(ECGM) (Promocell), that contained 0.4% ECGS/H, 2% Fetal Calf Serum, 0.1ng/ml Epidermal Growth Factor, 1 μg/ml Hydrocortison, 1 ng/ml basicFibroblast Factor and 1% penicillin/streptomycin (Gibco). 7 to 8 hlater, 50 μl starving medium (ECGM without supplements containing 0.5%heat inactivated FCS and 1% penicillin/streptomycin) was added to thecells and the cells were starved for 15 to 16 hours. 3 fold Serialdilutions of anti-VEGF scFvs (0.023-150 nM) and one of thefollowing—recombinant human VEGF₁₆₅ (0.08 nM), recombinant mouse VEGF₁₆₄(0.08 nM), or recombinant rat VEGF₁₆₄ (0.3 nM)—were prepared in starvingmedium and preincubated for 30-60 min at room temperature. The differentconcentrations of VEGFs were used to compensate for their differentrelative biological activities. Concentrations that stimulate submaximalVEGF induced proliferation (EC₉₀) were used. 100 μl of the mixtures wereadded to the 96-well tissue-culture plates containing the HUVECsuspension and incubated for 4 days in a 37° C./5% CO₂ humifiedincubator. Proliferation of HUVECs was assessed by measuring absorbanceat 450 nm (620 nm used as reference wavelength) after addition of 20μl/well WST-1 cell proliferation reagent (Roche) using a Sunrisemicroplate reader (Tecan). Data were analyzed using a 4-parameterlogistic curve-fit, and the concentration of anti-VEGF scFvs required toinhibit HUVEC proliferation by 50% (EC₅₀) was derived from inhibitioncurves.

The exemplary potency of lead candidates or their derivatives, measuredby HUVEC assays, is listed in Table 7. Further, the inhibition ofhVEGF₁₆₅-induced HUVEC proliferation by one derivative of leadcandidates, 578minmax, is exemplified in FIG. 9. EC50 of 578minmax forinhibition of hVEGF₁₆₅-induced cell-proliferation is determined to be0.06 nM (FIG. 9). The potency of 578minmax as a VEGF inhibitor is about1.6 times better compared to Lucentis. The inhibition of mouse or ratVEGF₁₆₄-induced HUVEC proliferation by 578minmax is also exemplified inFIG. 10. EC50 of 578minmax for inhibition of mouse and rat VEGF₁₆₄induced cell-proliferation is 0.06 nM and 0.07 nM, respectively (FIG.10). Thus, mouse and rat VEGF are equipotent to human VEGF for beinginhibited by the exemplary derivative (578minmax). Also in thisexperiment, Lucentis does not inhibit proliferation induced by rodentVEGF.

Example 6 Effects of Anti-VEGF scFvs on hVEGF₁₆₅ Induced VascularPermeability in Hairless Guinea Pigs

In this example, the effect of anti-VEGF scFvs on human VEGF₁₆₅ inducedvascular permeability was assessed in guinea pigs using the Miles assay.Thirty application sites per animal were marked on the dorsum ofhairless male guinea pigs using a permanent marker. On the treatment dayeach animal was administered intravenously with 1 ml of a 1% Evans bluedye solution under general anesthesia. One hour after dye injection, 0.1ml of test solution containing 2.61 nM recombinant human VEGF₁₆₅(PeproTech EC Ltd.) and various concentrations of anti-VEGF scFvs (0 nM,0.085 nM, 0.256 nM, 0.767 nM, 2.3 nM, 6.9 nM, 20.7 nM, 62.1 nM; n=7animals per test item) was injected in triplicate into the marks on thedorsum (3 injections per concentration of test item). Injections of PBSserved as a negative control in all animals. As an additional control,6.9 nM Lucentis (Novartis) was injected in all animals.

One hour after injection of the test solutions, the animals wereeuthanized, and the pelts were collected, cleaned, and photographeddigitally using incident and transmitted light. The area of Evans Bluedye that extravasated into the injection sites was evaluated usingImageJ. For each animal, anti-VEGF scFv concentration versus area of dyeleakage was analyzed using a 4-parameter logistic curve fit. Theconcentration of anti-VEGF scFvs required to inhibit vascular leakage by50% (EC₅₀) was derived from inhibition curves.

The experiment protocol is exemplified in FIG. 11. Also, the efficacy ofscFv candidates, ESBA903 (578minmax) and 802 (511max), in inhibiting thehVEGF was illustrated in FIG. 11, represented by different sizes ofareas containing the Evans Blue dye leaked from vascular system intoskin. The efficacy data for 903 and 802 are shown in FIG. 12. At 6.9 nM,903 and 802 showed stronger inhibition of VEGF induced vascular leakageinto the skin compared to Lucentis in all animals tested (FIG. 12).

Example 7 Effects of Topical Anti-VEGF scFvs Treatment on hVEGF₁₆₅Induced Retinal Vascular Leakage in Rats

In this example, topical efficacy of 578minmax is demonstarted using amodified Miles assay. These modifications include, for example, premixedstudy with intravitreal injections and topical application of scFvs.

Premixed different concentrations of anti-VEGF scFv (10, 3, and 1 foldmolar excess over VEGF) and VEGF (500 ng) were applied via a singleintravitreal injection. Avastin (Roche) (10, 3, and 1 fold molar excessover VEGF) was used as a positive control. Vehicle for 578minmax(Citrate Buffer, 20 mM Na-Citrate, 125 mM NaCl, pH 7) was used asnegative control. As illustrated in FIG. 13, premixing with hVEGF₁₆₅faciliated 578minmax (ESBA903) to completely inhibit hVEGF-inducedretinal vascular permeability. In this experiment, the inhibitory effectof 578minmax (ESBA903) was more significant compared to Avastin.

For topical application, five days before VEGF stimulation, adultSprague-Dawley rats received 578minmax (1%=10 mg/ml) via bilateraltopical dosing qid (4 drops/day) till perfusion day (Day 6). Vehicle for578minmax (topical dosing) and Alcon RTKi (10 mg/kg/d, oral gavage) wereused as negative and positive controls.

On Day 5, rats are anesthetized and their pupils are dilated. Allanimals receive intravitreal injections of 500 ng hrVEGF (10 μl) in botheyes. Following 24 hours post-injection of VEGF, intravenous infusion of3% Evans blue dye is performed on all animals during general anesthesia.After the dye has circulated for 90 minutes, the rats are euthanized.Blood samples are taken, then the rats are perfused with sterile salinesolution, then both eyes of each rat are immediately enucleated and theretinas harvested using a surgical microscope. For both retina andplasma samples, 60 μL of supernatant is used to measure the Evans bluedye absorbance (ABS) with a spectrophotometer at 620/740 nm. Theblood-retinal barrier breakdown and subsequent retinal vascularpermeability as measured by dye absorbance are calculated asmeans±s.e.m. of net ABS/wet weight/plasma ABS. One way ANOVA is used todetermine an overall difference between treatment means, where P 0.05 isconsidered significant. As exemplified in FIG. 14, the topicaladministration (5 days of pretreatment, 4 drops per day) of 578minmax(903) significantly inhibited hVEGF-induced retinal vascularpermeability. This is the first demonstration of a topically effectiveantibody useful for the treatment of intraocular disease.

EQUIVALENTS

Numerous modifications and alternative embodiments of the presentinvention will be apparent to those skilled in the art in view of theforegoing description. Accordingly, this description is to be construedas illustrative only and is for the purpose of teaching those skilled inthe art the best mode for carrying out the present invention. Details ofthe structure may vary substantially without departing from the spiritof the invention, and exclusive use of all modifications that comewithin the scope of the appended claims is reserved. It is intended thatthe present invention be limited only to the extent required by theappended claims and the applicable rules of law.

All literature and similar material cited in this application,including, patents, patent applications, articles, books, treatises,dissertations, web pages, figures and/or appendices, regardless of theformat of such literature and similar materials, are expresslyincorporated by reference in their entirety. In the event that one ormore of the incorporated literature and similar materials differs fromor contradicts this specification, including defined terms, term usage,described techniques, or the like, this specification controls.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the present inventions have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present inventions encompass various alternatives, modifications,and equivalents, as will be appreciated by those of skill in the art.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made without departing fromthe scope of the appended claims. Therefore, all embodiments that comewithin the scope and spirit of the following claims and equivalentsthereto are claimed.

SEQUENCE LISTING SEQ ID NO: 1 Peptide Immunogen KFMDVYQRSYCVL sequences: SEQ ID NO. 72: 60EVVMAQTPASVEAAVGGTVTIKCQASQSISSYLSWYQQKPGQPPKLLIYKASTLASGVPSRFKGSRSGTEYTLTISDLECADAATYYCQSNYGGSSSDYG NPFGGGTEAVVKSEQ ID NO. 73: 435 AFELTQTPSSVEAAVGGTVTIKCQASQSIGSSLAWYQQKPGQRPKLLIYTAANLASGVPSRFRGSRSGAAFTLTISDLECADAATYYCQNFATSDTVTFG GGTEVVVTSEQ ID NO. 74: 453 AVVLTQTPSPVSAAVGGTVSISCQSSQSVWNNNRLAWFQQKSGQPPKLLIYYASTLASGVPSRFKGSGSGTEFTLTISDVQCDDAATYYCAGGYSSTSDN TFGGGTEVVVKSEQ ID NO. 75: 375 DIVMTQTPASVEATVGGTITINCQASENINIWLSWYQQKPGQPPKLLIYQASKLASGVPSRFKGSGSGTQFTLTISDLECADAATYYCQNNYSYNRYGAP FGGGTEVVVKSEQ ID NO. 76: 610 DVVMTQTPASVSEPVGGTVTIKCQASQSISSWLSWYQQKPGQPPKLLIYQASTLASGVPPRSSGSGSGTEYTLTISDLECADAATYFCQNNYGFRSYGGA FGGGTEVVVKSEQ ID NO. 77: 578 DVVMTQTPSSVSAAVGDTVTINCQASEIIHSWLAWYQQKPGQPPKLLIYLASTLASGVPSRFKGSGSGTQFTLTISDLECADAAIYYCQNVYLASTNGAN FGGGTEVVVKSEQ ID NO. 78: 534 DVVMTQTPSSVSAAVGDTVTIKCQASQSINIWLSWYQQKSGQPPKLLVYKESTLASGVPSRFRGSGSGTQFTLTISDLECADAATYYCQNNYDSGNNGFP FGGGTEVVVKSEQ ID NO. 79: 567 DVVMTQTPSSVSAAVGDTVTINCQADQSIYIWLSWYQQKPGQPPKLLIYKASTLESGVPSRFKGSGSGTQFTLTISDLECADAATYYCQNNAHYSTNGGT FGGGTEVVVKSEQ ID NO. 80: 509 DVVMTQTPSSVSAAVGDTVTIKCQASQNIRIWLSWYQQKPGQPPKLLIYKASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQNNAHYSTNGGT FGGGTEVVVKSEQ ID NO. 81: 511 EVVMTQTPASVEAAVGGTVTIKCQASQSINIWCSWYQQKPGHPPKLLIYRASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQANYAYSAGYGA AFGGGTEVVVKSEQ ID NO. 82: 60 min EIVMTQSPSTLSASVGDRVIITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQSNYGGSSSDYG NPFGQGTKLTVLGSEQ ID NO. 83: 435 minEIVMTQSPSTLSASVGDRVIITCQASQSIGSSLAWYQQKPGKAPKLLIYTAANLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNFATSDTVTFG QGTKLTVLGSEQ ID NO. 84: 453 minEIVMTQSPSTLSASVGDRVIITCQSSQSVWNNNRLAWYQQKPGKAPKLLIYYASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGGYSSTSDN TFGQGTKLTVLGSEQ ID NO. 85: 375 minEIVMTQSPSTLSASVGDRVIITCQASENINIWLSWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNNYSYNRYGAP FGQGTKLTVLGSEQ ID NO. 86: 610 minEIVMTQSPSTLSASVGDRVIITCQASQSISSWLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNNYGFRSYGGA FGQGTKLTVLGSEQ ID NO. 87: 578 minEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGAN FGQGTKLTVLGSEQ ID NO. 88: 534 minEIVMTQSPSTLSASVGDRVIITCQASQSINIWLSWYQQKPGKAPKLLIYKESTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNNYDSGNNGFP FGQGTKLTVLGSEQ ID NO. 89: 567 minEIVMTQSPSTLSASVGDRVIITCQADQSIYIWLSWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNNAHYSTNGGT FGQGTKLTVLGSEQ ID NO. 90: 509 minEIVMTQSPSTLSASVGDRVIITCQASQNIRIWLSWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNNAHYSTNGGT FGQGTKLTVLGSEQ ID NO. 91: 511 minEIVMTQSPSTLSASVGDRVIITCQASQSINIWCSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQANYAYSAGYGA AFGQGTKLTVLGSEQ ID NO. 92: 578 min_Pref substEIVLTQSPSSLSASVGDRVTITCQASEIIHSWLAWYQQRPGKAPKLLISLASTLASGVPSRFSGSGSGTDFTFTISSLQPEDFAVYYCQNVYLASTNGAN FGQGTKVEIKRSEQ ID NO. 93: 60 max EIVMTQSPSTLSASVGDRVIITCQASQSISSYLSWYQQKPGKAPKLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSNYGGSSSDYG NPFGQGTKLTVLGSEQ ID NO. 94: 435 maxEIVMTQSPSTLSASVGDRVIIKCQASQSIGSSLAWYQQKPGKAPKLLIYTAANLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNFATSDTVTFG QGTKLTVLGSEQ ID NO. 95: 453 maxEIVMTQSPSTLSASVGDRVIITCQSSQSVWNNNRLAWYQQKPGKAPKLLIYYASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCAGGYSSTSDN TFGQGTKLTVLGSEQ ID NO. 96: 375 maxEIVMTQSPSTLSASVGDRVIITCQASENINIWLSWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCQNNYSYNRYGAP FGQGTKLTVLGSEQ ID NO. 97: 610 maxEIVMTQSPSTLSASVGDRVIITCQASQSISSWLSWYQQKPGKAPKLLIYQASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNNYGFRSYGGA FGQGTKLTVLGSEQ ID NO. 98: 578 maxEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCQNVYLASTNGAN FGQGTKLTVLGSEQ ID NO. 99: 534 maxEIVMTQSPSTLSASVGDRVIITCQASQSINIWLSWYQQKPGKAPKLLIYKESTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNNYDSGNNGFP FGQGTKLTVLGSEQ ID NO. 100: 567 maxEIVMTQSPSTLSASVGDRVIITCQADQSIYIWLSWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCQNNAHYSTNGGT FGQGTKLTVLGSEQ ID NO. 101: 509 maxEIVMTQSPSTLSASVGDRVIITCQASQNIRIWLSWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNNAHYSTNGGT FGQGTKLTVLGSEQ ID NO. 102: 511 maxEIVMTQSPSTLSASVGDRVIITCQASQSINIWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQANYAYSAGYGA AFGQGTKLTVLGSEQ ID NO. 103: 578 max_Pref substEIVMTQSPSSLSASVGDRVTITCQASEIIHSWLAWYQQRPGKAPKLLISLASTLASGVPSRFSGSGSGTQFTFTISSLQPEDFAVYYCQNVYLASTNGAN FGQGTKVEIKRSEQ ID NO. 104: 578 min VL: E1DDIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGAN FGQGTKLTVLGSEQ ID NO. 105: 578 min VL: I2VEVVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGAN FGQGTKLTVLGSEQ ID NO. 106: 511 min VL: C41LEIVMTQSPSTLSASVGDRVIITCQASQSINIWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQANYAYSAGYGA AFGQGTKLTVLGVH sequences: SEQ ID NO. 107: 60-11-4QSLEESGGDLVKPGASLTLTCTASGFPFSSGYWVCWVRQAPGKGLEWIACIYAGSSGSTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGNNYYIYTDGGYAYAGLELWGPGILVTVSS SEQ ID NO. 108: 60-11-6QSLEESGGDLVKPGASLTLTCTASGFSFSSGYWICWVRQAPGKGLEWIACIYAGSSGSTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGNNYYIYTDGGYAYAGLELWGPGILVTVSS SEQ ID NO. 109: 435QSLEESGGDLVQPGASLTLTCKVSGFSLNTNYWMCWVRQAPGKGLEWIGCMYTGSYNRAYYASWAKGRFTSSKTSSTTVTLEMTSLTAADTATYFCAKGS NWYSDLWGPGTLVTVSSSEQ ID NO. 110: 453 QERLVESGGGLVQPEGSLTLTCKASGFSFSRSYYIYWVRQAPGKGLEWIACIDAGSSGILVYANWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDASYGVDSFMLPLWGPGTLVTVSS SEQ ID NO. 111: 375QSLEESGGGLVQPEGSLTLTCKASGFSFTTTDYMCWVRQAPGKGLEWIGCILAGDGSTYYANWAKGRFTGSKTSSTTVDLKMTGLTAADTATYFCARSDP ASSWSFALWGPGTLVTVSSSEQ ID NO. 112: 610 QSLEESGGRLVTPGTPLTLTCTASGIDFSGAYYMGWVRQAPGKGLEWIGYIDYDGDRYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARSDYSS GWGTDIWGPGTLVTVSLSEQ ID NO. 113: 578 QSVEESGGRLVTPGTPLTLTCTASGFSLTDYYYMTWVRLAPGKGLEYIGFIDPDDDPYYATWAKGRFTISRTSTTVNLKMTSPTTEDTATYFCAGGDHNS GWGLDIWGPGTLVTVSLSEQ ID NO. 114: 534 QSLEESGGRLVTPGTPLTLTCTASGFSLSYYYMSWVRQAPGKGLEWIGIIGPGDYTDYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCGRGDDNSG WGEDIWGPGTLVTVSLSEQ ID NO. 115: 567 QSVEESGGRLVTPGAPLTLTCSVSGFSLSDYYMCWVRQAPGKGLQWIGCLDYFGSTDDASWAKGRFTISKTSTAVDLKITSPTTEDTATYFCARTDDSRG WGLNIWGPGTLVTVSLSEQ ID NO. 116: 509 QSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMCWVRQAPGKGLEWIGCLDYVGDTDYASWAKGRFTISKASTTVDLKITSLTTEDTATYFCARTDDSRG WGLNIWGPGTLVTVSLSEQ ID NO. 117: 511 QSVEESGGRLVTPGTPLTLTCTVSGFSLNTYYMNWVRQAPGKGLEWIGIIAPDDTTYYASWAKSRSTITRDTNENTVTLKMTSLTTEDTATYFCARSGDT TAWGADIWGPGTLVTVSLSEQ ID NO. 118: 60-11-4 minEVQLVESGGGLVQPGGSLRLSCAASGFPFSSGYWVCWVRQAPGKGLEWVSCIYAGSSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGNNYYIYTDGGYAYAGLELWGQGTLVTVSS SEQ ID NO. 119: 60-11-6 minEVQLVESGGGLVQPGGSLRLSCAASGFSFSSGYWICWVRQAPGKGLEWVSCIYAGSSGSTYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGNNYYIYTDGGYAYAGLELWGQGTLVTVSS SEQ ID NO. 120: 435 minEVQLVESGGGLVQPGGSLRLSCAASGFSLNTNYWMCWVRQAPGKGLEWVSCMYTGSYNRAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GSNWYSDLWGQGTLVTVSSSEQ ID NO. 121: 453 minEVQLVESGGGLVQPGGSLRLSCAASGFSFSRSYYIYWVRQAPGKGLEWVSCIDAGSSGILVYANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDASYGVDSFMLPLWGQGTLVTVSS SEQ ID NO. 122: 375 minEVQLVESGGGLVQPGGSLRLSCAASGFSFTTTDYMCWVRQAPGKGLEWVSCILAGDGSTYYANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKS DPASSWSFALWGQGTLVTVSSSEQ ID NO. 123: 610 minEVQLVESGGGLVQPGGSLRLSCAASGIDFSGAYYMGWVRQAPGKGLEWVSYIDYDGDRYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSD YSSGWGTDIWGQGTLVTVSSSEQ ID NO. 124: 578 minEVQLVESGGGLVQPGGSLRLSCAASGFSLTDYYYMTWVRQAPGKGLEWVSFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 125: 534 minEVQLVESGGGLVQPGGSLRLSCAASGFSLSYYYMSWVRQAPGKGLEWVSIIGPGDYTDYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDD NSGWGEDIWGQGTLVTVSSSEQ ID NO. 126: 567 minEVQLVESGGGLVQPGGSLRLSCAASGFSLSDYYMCWVRQAPGKGLEWVSCLDYFGSTDDASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTDD SRGWGLNIWGQGTLVTVSSSEQ ID NO. 127: 509 minEVQLVESGGGLVQPGGSLRLSCAASGFSLSSYYMCWVRQAPGKGLEWVSCLDYVGDTDYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTDD SRGWGLNIWGQGTLVTVSSSEQ ID NO. 128: 511 minEVQLVESGGGLVQPGGSLRLSCAASGFSLNTYYMNWVRQAPGKGLEWVSIIAPDDTTYYASWAKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGD TTAWGADIWGQGTLVTVSSSEQ ID NO. 129: 578 min_Pref substsubstQVQLVQTGGGLVQPGGSLRLSCAASGFSLTDYYYMTWVRQAPGKGLEWVSFIDPDDDPYYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTALYYCAKGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO. 130: 60-11-4 maxEVQLVESGGGLVQPGGSLRLSCTASGFPFSSGYWVCWVRQAPGKGLEWVGCIYAGSSGSTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGNNYYIYTDGGYAYAGLELWGQGTLVTVSS SEQ ID NO. 131: 60-11-6 maxEVQLVESGGGLVQPGGSLRLSCTASGFSFSSGYWICWVRQAPGKGLEWVGCIYAGSSGSTYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGNNYYIYTDGGYAYAGLELWGQGTLVTVSS SEQ ID NO. 132: 435 maxEVQLVESGGGLVQPGGSLRLSCKVSGFSLNTNYWMCWVRQAPGKGLEWVGCMYTGSYNRAYYASWAKGRFTSSKDTSKNTVYLQMNSLRAEDTAVYYCAK GSNWYSDLWGQGTLVTVSSSEQ ID NO. 133: 453 maxEVQLVESGGGLVQPGGSLRLSCKASGFSFSRSYYIYWVRQAPGKGLEWVGCIDAGSSGILVYANWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGDASYGVDSFMLPLWGQGTLVTVSS SEQ ID NO. 134: 375 maxEVQLVESGGGLVQPGGSLRLSCKASGFSFTTTDYMCWVRQAPGKGLEWVGCILAGDGSTYYANWAKGRFTGSKDTSKNTVYLQMNSLRAEDTAVYYCARS DPASSWSFALWGQGTLVTVSSSEQ ID NO. 135: 610 maxEVQLVESGGGLVQPGGSLRLSCTASGIDFSGAYYMGWVRQAPGKGLEWVGYIDYDGDRYYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARSD YSSGWGTDIWGQGTLVTVSSSEQ ID NO. 136: 578 maxEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 137: 534 maxEVQLVESGGGLVQPGGSLRLSCTASGFSLSYYYMSWVRQAPGKGLEWVGIIGPGDYTDYASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGDD NSGWGEDIWGQGTLVTVSSSEQ ID NO. 138: 567 maxEVQLVESGGGLVQPGGSLRLSCSVSGFSLSDYYMCWVRQAPGKGLEWVGCLDYFGSTDDASWAKGRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARTDD SRGWGLNIWGQGTLVTVSSSEQ ID NO. 139: 509 maxEVQLVESGGGLVQPGGSLRLSCTASGFSLSSYYMCWVRQAPGKGLEWVGCLDYVGDTDYASWAKGRFTISKDASKNTVYLQMNSLRAEDTAVYYCARTDD SRGWGLNIWGQGTLVTVSSSEQ ID NO. 140: 509 maxIIEVQLVESGGGLVQPGGSLRLSCTASGFSLSSYYMSWVRQAPGKGLEWVGILDYVGDTDYASWAKGRFTISKDASKNTVYLQMNSLRAEDTAVYYCARTDD SRGWGLNIWGQGTLVTVSSSEQ ID NO. 141: 511 maxEVQLVESGGGLVQPGGSLRLSCTVSGFSLNTYYMNWVRQAPGKGLEWVGIIAPDDTTYYASWAKSRSTISRDTSKNTVYLQMNSLRAEDTAVYYCARSGD TTAWGADIWGQGTLVTVSSSEQ ID NO. 142: 578 max_Pref substsubstQVQLVQTGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTALYYCAGGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO. 143: 567 minDHPEVQLVESGGGSVQPGGSLRLSCAASGFSLSDYYMCWVRQAPGKGLEWVSCLDYFGSTDDASWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAKTDD SRGWGLNIWGQGTTVTVSSSEQ ID NO. 144: 578 maxDHPEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSSEQ ID NO. 145: 511 maxDHPEVQLVESGGGSVQPGGSLRLSCTVSGFSLNTYYMNWVRQAPGKGLEWVGIIAPDDTTYYASWAKSRSTISRDTSKNTVYLQMNSLRAEDTATYYCARSGD TTAWGADIWGQGTTVTVSSSEQ ID NO. 146: 578 max_Pref subst_DHPQVQLVQTGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSSEQ ID NO. 147: 578 max VH: E1_VQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGDH NSGWGLDIWGQGTLVTVSSSEQ ID NO. 148: 578 max VH: V2QEQQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 149: 578 max VH: Q46LEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRLAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 150: 578 max VH: W54YEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEYVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 151: 578 max VH: V55IEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWIGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 152: 578 max VH: D83AEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRATSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 153: 578 max VH: N87AEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKATVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 154: 578 max VH: Y105FEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYFCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 155: 578 max VH: D83_EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRTSKNTVYLQMNSLRAEDTAVYYCAGGDH NSGWGLDIWGQGTLVTVSSSEQ ID NO. 156: 578 max VH: N87_EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKTVYLQMNSLRAEDTAVYYCAGGDH NSGWGLDIWGQGTLVTVSSSEQ ID NO. 157: 578 max VH: T84NEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 158: 578 max VH: V89LEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTLYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 159: 578 max VH: V89AEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTAYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 160: 578 maxDHP VH: T84NEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSSEQ ID NO. 161: 578 maxDHP VH: V89LEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTLYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSSEQ ID NO. 162: 578 maxDHP VH: V89AEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTAYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSSEQ ID NO. 163: 578 max VH: T84N, V89AEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 164: 578 max VH: T84N, V89LEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGD HNSGWGLDIWGQGTLVTVSSSEQ ID NO. 165: 578 maxDHP VH: T84N, V89AEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTAYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSSEQ ID NO. 166: 578 maxDHP VH: T84N, V89LEVQLVESGGGSVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAGGD HNSGWGLDIWGQGTTVTVSSframework sequences (X residues are CDRinsertion sites and may be any naturallyoccurring amino acid. At least 3 and up to50 amino acids can be present): SEQ ID NO. 167: Variable light chainFW1.4 and rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50)WYQQKPGKAPKLLIY(X)_(n=3-50) GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)_(n=3-50)FGQGTKLT VLG SEQ ID NO. 168: Variable light chain rFW1.4 variant 2 (v2)EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50) WYQQKPGKAPKLLIY(X)_(n=3-50)GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)_(n=3-50) FGQGTKLT VLGSEQ ID NO. 169: Variable heavy chain FW1.4EVQLVESGGGLVQPGGSLRLSCAAS(X)_(n=3-50) WVRQAPGKGLEWVS (X)_(n=3-50)RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(X)_(n=3-50) WGQGTL VTVSSSEQ ID NO. 170: Variable heavy chain rFW1.4EVQLVESGGGLVQPGGSLRLSCTAS(X)_(n=3-50) WVRQAPGKGLEWVG(X)_(n=3-50)RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=3-50) WGQGTLV TVSSEQ ID NO. 171: Variable heavy chain rFW1.4 variant 2 (v2)EVQLVESGGGLVQPGGSLRLSCTVS(X)_(n=3-50) WVRQAPGKGLEWVG(X)_(n=3-50)RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X)_(n=3-50) WGQGTLVTVSSScFv framework sequences: SEQ ID NO. 172: FW1.4EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50) WYQQKPGKAPKLLIY(X)_(n=3-50)GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)_(n=3-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAAS (X)_(n=3-50)WVRQAPGKGLEWVS(X)_(n=3-50) RFTISRDNSKNTLYLQMNSLRAEDTA VYYCAK(X)_(n=3-50)WGQGTLVTVSS SEQ ID NO. 173: rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50)WYQQKPGKAPKLLIY(X)_(n=3-50) GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=3-50)FGQGTKLTVLG GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS (X)_(n=3-50)WVRQAPGKGLEWVG(X)_(n=3-50) RFTISRDTSKNTVYLQMNS LRAEDTAVYYCAR(X)_(n=3-50)WGQGTLVTVSS SEQ ID NO. 174: rFW1.4 variant 2 (v2)EIVMTQSPSTLSASVGDRVIITC(X)_(n=3-50) WYQQKPGKAPKLLIY(X)_(n=3-50)GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X)_(n=3-50) FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCTVS(X)_(n=3-50)WVRQAPGKGLEWVG(X)_(n=3-50) RFTISKDTSKNTVYLQMNSLR AEDTAVYYCAR(X)_(n=3-50)WGQGTLVTVSS ScFv anti-VEGF sequences: SEQ ID NO. 175: 435_maxEIVMTQSPSTLSASVGDRVIITCQASQSIGSSLAWYQQKPGKAPKLLIYTAANLASGVPSRFSGSRSGAEFTLTISSLQPDDFATYYCQNFATSDTVTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKASGFSLNTNYWMCWVRQAPGKGLEWVGCMYTGSYNRAYYASWAKGRFTSSKDTSKNTVYLQMNSLRAEDTAVYYCAKGSNWYSDLWGQGTLVTVSS SEQ ID NO. 176: 511_maxEIVMTQSPSTLSASVGDRVIITCQASQSINIWLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQANYAYSAGYGAAFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLNTYYMNWVRQAPGKGLEWVGIIAPDDTTYYASWAKSRSTISRDTSKNTVYLQMNSLRAEDTAVYYCARSGDTTAWGADIWGQGTLVTVS SSEQ ID NO. 177: 567_minEIVMTQSPSTLSASVGDRVIITCQADQSIYIWLSWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNNAHYSTNGGTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSDYYMCWVRQAPGKGLEWVSCLDYFGSTDDASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKTDDSRGWGLNIWGQGTLVTVSSSEQ ID NO. 178: 578 minEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLTDYYYMTWVRQAPGKGLEWVSFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDHNSGWGLDIWGQGTLVTVS SSEQ ID NO. 179: 578 maxEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGTQFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVS SSEQ ID NO. 180: 578 minmax (ESBA903)EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVS SSEQ ID No. 181 linker GGGGSGGGGSGGGGSGGGGS

1-63. (canceled)
 64. A recombinant immunobinder comprising a variableheavy chain (VH) and/or a variable light chain (VL) which neutralizeshuman VEGF and comprises rabbit CDRs.
 65. The immunobinder of claim 64being a chimeric immunobinder.
 66. The immunobinder of claim 64 beinghumanized.
 67. The immunobinder of claim 64, comprising at least threeCDRs, each having at least 80% similarity to a consensus sequence of thegroup consisting of SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 37, SEQ IDNO: 49, SEQ ID NO: 60 and SEQ ID NO:
 71. 68. The immunobinder of claim67, wherein: a. the VH comprises CDRs of the group consisting of SEQ IDNO: 2, SEQ ID NO: 15 and SEQ ID NO: 27 and wherein the VL comprises CDRsof the group consisting of SEQ ID NO: 38, SEQ ID NO: 50 and SEQ ID NO:61; b. the VH comprises CDRs of the group consisting of SEQ ID NO: 3,SEQ ID NO: 15 and SEQ ID NO: 27 and the VL comprises CDRs of the groupconsisting of SEQ ID NO: 38, SEQ ID NO: 50 and SEQ ID NO: 61; c. the VHcomprises CDRs of the group consisting of SEQ ID NO: 4, SEQ ID NO: 16,SEQ ID NO: 28 and the VL comprises CDRs of the group consisting of SEQID NO: 39, SEQ ID NO: 51, and SEQ ID NO: 62; d. the VH comprises CDRs ofthe group consisting of SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 29 andthe VL comprises CDRs of the group consisting of SEQ ID NO: 40, SEQ IDNO: 52 and SEQ ID NO: 63; e. the VH comprises CDRs of the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 18 and SEQ ID NO: 30 and the VLcomprises CDRs of the group consisting of SEQ ID NO: 41, SEQ ID NO: 53and SEQ ID NO: 64; f. the VH comprises CDRs of the group consisting ofSEQ ID NO: 7, SEQ ID NO: 19 and SEQ ID NO: 31 and the VL comprises CDRsof the group consisting of SEQ ID NO: 42, SEQ ID NO: 54 and SEQ ID NO:65; g. the VH comprises CDRs of the group consisting of SEQ ID NO: 8,SEQ ID NO: 20 and SEQ ID NO: 32 and the VL comprises CDRs of the groupconsisting of SEQ ID NO: 43, SEQ ID NO: 55 and SEQ ID NO: 66; h. the VHcomprises CDRs of the group consisting of SEQ ID NO: 9, SEQ ID NO: 21and SEQ ID NO: 33 and the VL comprises CDRs of the group consisting ofSEQ ID NO: 44, SEQ ID NO: 56 and SEQ ID NO: 67; i. the VH comprises CDRsof the group consisting of SEQ ID NO: 10, SEQ ID NO: 22 and SEQ ID NO:34 and the VL comprises CDRs of the group consisting of SEQ ID NO: 45,SEQ ID NO: 57 and SEQ ID NO: 68; j. the VH comprises CDRs of (i) thegroup consisting of SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 35; (ii)the group consisting of SEQ ID NO: 11, SEQ ID NO: 25 and SEQ ID NO: 35;(iii) the group consisting of SEQ ID NO: 13, SEQ ID NO: 23 and SEQ IDNO: 35; or (iv) the group consisting of SEQ ID NO: 13, SEQ ID NO: 25 andSEQ ID NO: 35; and the VL comprises CDRs of the group consisting of SEQID NO: 46, SEQ ID NO: 58 and SEQ ID NO: 69; k. the VH comprises CDRs ofthe group consisting of SEQ ID NO: 12, SEQ ID NO: 24 and SEQ ID NO: 36and wherein the VL comprises (i) CDRs of the group consisting of SEQ IDNO: 47, SEQ ID NO: 59 and SEQ ID NO: 70; or (ii) CDRs of the groupconsisting of SEQ ID NO: 48, SEQ ID NO: 59 and SEQ ID NO:
 70. 69. Theimmunobinder of claim 64 comprising a heavy chain variable regionvariable region framework sequence having at least 80% sequence identityto the sequence of SEQ ID NO:
 169. 70. The immunobinder of claim 69,wherein the heavy chain variable region framework is SEQ ID NO: 170 orSEQ ID NO:
 171. 71. The immunobinder of claim 68, having deletions atpositions 1, 83 or 87 of the heavy chain variable region according tothe AHo numbering system.
 72. The immunobinder of claim 68, having asubstitution in at least one of positions 2, 12, 24, 25, 46, 54, 55, 83,84, 87, 89, 103, 105, 108 or 144 in the heavy chain variable regionaccording to the AHo numbering system.
 73. The immunobinder of claim 72,wherein said substitutions are selected from the group consisting of:(a) Glutamine at position 2; (b) Serine at position 12; (c) Threonine atposition 24; (d) Valine at position 25; (e) Leucine at position 46; (f)Tyrosine at position 54; (g) Isoleucine at position 55; (h) Alanine atposition 83; (i) Asparagine at position 84 (j) Alanine or Leucine atposition 87; (k) Alanine or Leucine at position 89; (l) Serine orThreonine at position 103; (m) Phenylalanine at position 105; (n)Arginine at position 108; and (o) Serine or Threonine at position 144.74. The immunobinder of claim 64, comprising a light chain variableregion framework sequence having at least 85% sequence identity to thesequence of SEQ ID NO:
 167. 75. The immunobinder of claim 74, whereinthe light chain variable region framework is SEQ ID NO: 167 or SEQ IDNO:
 168. 76. The immunobinder of claim 68, having a substitution in atleast one of positions 1, 2, or 15 of the light chain variable regionaccording to the AHo numbering system.
 77. The immunobinder of claim 76,wherein said substitutions are selected from the group consisting of:(a) Aspartate at position 1; (b) Valine at position 2; and (c) Threonineat position
 15. 78. The immunobinder of claim 64, comprising: a. a heavychain variable region comprising an amino acid sequence at least 80%similar to SEQ ID NO: 107 and a light chain variable region at least 80%similar to SEQ ID NO: 72; b. a heavy chain variable region comprising anamino acid sequence at least 80% similar to SEQ ID NO: 109 and a lightchain variable region at least 80% similar to SEQ ID NO: 73; c. a heavychain variable region comprising an amino acid sequence at least 80%similar to SEQ ID NO: 110 and a light chain variable region at least 80%similar to SEQ ID NO: 74; d. a heavy chain variable region comprising anamino acid sequence at least 80% similar to SEQ ID NO: 111 and a lightchain variable region at least 80% similar to SEQ ID NO: 75; e. a heavychain variable region comprising an amino acid sequence at least 80%similar to SEQ ID NO: 112 and a light chain variable region at least 80%similar to SEQ ID NO: 76; f. a heavy chain variable region comprising anamino acid sequence at least 80% similar to SEQ ID NO: 113 and a lightchain variable region at least 80% similar to SEQ ID NO: 77; g. a heavychain variable region comprising an amino acid sequence at least 80%similar to SEQ ID NO: 114 and a light chain variable region at least 80%similar to SEQ ID NO: 78; h. a heavy chain variable region comprising anamino acid sequence at least 80% similar to SEQ ID NO: 115 and a lightchain variable region at least 80% similar to SEQ ID NO: 79; i. a heavychain variable region comprising an amino acid sequence at least 80%similar to SEQ ID NO: 116 and a light chain variable region at least 80%similar to SEQ ID NO: 80; j. a heavy chain variable region comprising anamino acid sequence at least 80% similar to SEQ ID NO: 117 and a lightchain variable region at least 80% similar to SEQ ID NO:
 81. 79. Theimmunobinder of claim 64, having at least 80% identity to SEQ ID NO:176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179 or SEQ ID NO: 180.80. An immunobinder, which binds with an affinity of at least 10⁷ M⁻¹ toan epitope on human VEGF recognized by the immunobinder of claim
 64. 81.The immunobinder of claim 64 which specifically binds to human andrat/mouse VEGF.
 82. The immunobinder of claim 64 which is an antibody,scFv, Fab or Dab.
 83. A composition comprising the immunobinder of claim64, and a pharmaceutically acceptable carrier.
 84. An isolated nucleicacid molecule encoding the immunobinder of claim
 64. 85. An expressionvector comprising the nucleic acid molecule of claim
 84. 86. A host cellcomprising the expression vector of claim
 85. 87. A method of treatingor preventing a human VEGF-mediated disease in a subject, comprisingadministering to a subject an immunobinder of claim 64 such that thesubject is treated for a VEGF-mediated disease.
 88. The method of claim87, wherein the VEGF-mediated disease is selected from the groupconsisting of age-related macular degeneration, neovascular glaucoma,diabetic retinopathy, retinopathy of prematurity, retrolentalfibroplasia, breast carcinomas, lung carcinomas, gastric carcinomas,esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovariancarcinomas, the comas, arrhenoblastomas, cervical carcinomas,endometrial carcinoma, endometrial hyperplasia, endometriosis,fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngealcarcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma,melanoma, skin carcinomas, hemangioma, cavernous hemangioma,hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma,glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma,neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas,urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cellcarcinoma, prostate carcinoma, abnormal vascular proliferationassociated with phakomatoses, edema (such as that associated with braintumors), Meigs' syndrome, rheumatoid arthritis, psoriasis andatherosclerosis.
 89. A hybridoma producing an antibody comprising anyone of the amino acid sequences set forth in SEQ ID NOs: 60-166 and inSEQ ID NOs: 175-180.
 90. An immunogen suitable for raising oridentifying an immunobinder that is cross reactive with rat/mouse andhuman VEGF or a portion thereof.
 91. The immunogen of claim 90 havingSEQ ID NO:
 1. 92. A method of making an immunobinder that is crossreactive with rat/mouse and human VEGF, wherein the method employs theimmunogen of claim
 91. 93. The composition of claim 83, formulated fortopical, intraocular, oral, nasal, rectal or parental administration.94. A method of treating a VEGF-mediated disease in a mammal, inparticular in a human, comprising administering to a subject thecomposition of claim 83 in a pharmaceutically acceptable dosage form bytopical, intraocular, intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intraarticular, intrasynovial,intrathecal, oral, or inhalation routes.